Editing Cleancap mRNA Invitro Transcription (section)

= Protocol for Co-transcriptional capping using CleanCap<sup>®</sup>&nbsp;Reagent AG from TriLink and HiScribe<sup>™</sup>&nbsp;T7 High Yield RNA Synthesis Kit from New England Biolabs<sup>®</sup> =

'''Reagents required:'''

1. NEB HiScribe<sup>™</sup>&nbsp;T7 High Yield RNA Synthesis Kit, NEB #&nbsp;&nbsp;[https://www.neb.com/products/e2040-hiscribe-t7-high-yield-rna-synthesis-kit https://www.neb.com/products/e2040-hiscribe-t7-high-yield-rna-synthesis-kit]&nbsp;E2040S&nbsp;<br/>2. TriLink CleanCap<sup>®</sup>&nbsp;Reagent AG, N-7113, or CleanCap<sup>®</sup>&nbsp;Reagent AG (3’ OMe), N-7413

https://www.trilinkbiotech.com/media/catalog/product/cache/c687aa7517cf01e65c009f6943c2b1e9/n/-/n-7413.png





{|
|-
! scope="row" | Extinction Coefficient
| data-th="Extinction Coefficient" | 30,539 Lmol<sup>-1</sup>cm<sup>-1</sup>&nbsp;at 255 nm
|-
! scope="row" | Molecular Formula
| data-th="Molecular Formula" | C<sub>33</sub>H<sub>45</sub>N<sub>15</sub>O<sub>24</sub>P<sub>4</sub>&nbsp;(free acid)
|-
! scope="row" | Molecular Weight
| data-th="Molecular Weight" | 1159.60
|-
! scope="row" | Salt Form
| data-th="Salt Form" | Na+
|-
! scope="row" | Concentration
| data-th="Concentration" | 100 mM
|-
! scope="row" | Buffer
| data-th="Buffer" | H<sub>2</sub>O
|-
! scope="row" | Recommended Storage
| data-th="Recommended Storage" | -20°C or below
|-
! scope="row" | Other Name(s)
| data-th="Other Name(s)" | CleanCap<sup>®</sup>Reagent AG (3' OMe) for co-transcriptional capping of mRNA, m7(3'OMeG)(5')ppp(5')(2'OMeA)pG
|}

10 nmole ==>11.6 mg

to make 100 mM, 11.6 mg + 100 ul H2O

to calculate more acculately, dilute 1000 fold, use nanodrop, 30,539 Lmol-1cm-1 at 255 nm

=== ARCA (m2<sup>7,3'-O</sup>GP<sub>3</sub>G or&nbsp;3′-O-Me-m7G(5′) ppp(5′)G ===

[[File:ARCA Cap Analog.png|RTENOTITLE]]

https://www.targetmol.com/compound/sb%20216641%20hydrochloride



=== CleanCap AG (3'OMe) ===

[[File:CleanCap AG (3'OMe)n-7413.png|RTENOTITLE]]

===  ===

[[File:ARCA Cap Analog.png|RTENOTITLE]]

=== Cap analogues ===

[[File:Cap annalogues.png|RTENOTITLE]]

==  ==

== [[File:Cleancap vs ARCA.png|RTENOTITLE]] ==

Pfizer

[[File:Phfizer GA cap.png|RTENOTITLE]]

== Trinucleotide cap1 analogue ==

((m2 7,3’-O)Gppp(m2’-O)ApG; TriLink)

==  ==

== UTP&nbsp; ==

Uridine 5'-Triphosphate, Sigma , 94370-1G, 1 g $288

[[File:Uridine-5'-Triphosphate.png|RTENOTITLE]]

Pseudouridine-5'-Triphosphate,&nbsp;Pseudo-UTP, 5-Ribosyl Uracil,&nbsp; N-1019-5,5 µmole $451

Pseudouridine (5-ribosyluracil) was the first modified ribonucleoside discovered. It is the most abundant natural modified RNA base, and has been deemed the "fifth nucleoside" in RNA. It can be found in structural RNAs, such as transfer, ribosomal and small nuclear RNA. Pseudouridine has been found to enhance base stacking and translation. Pseudouridine-5'-triphosphate (Pseudo-UTP) is used to impart desirable mRNA characteristics such as increased nuclease stability, increased translation or altered interaction of innate immune receptors with in vitro transcribed RNA. Pseudo-UTP, along with 5-methylcytidine-5'-triphosphate (5-methyl-CTP) has shown innate immune suppression in culture and in vivo while enhancing translation in recent publications. For example, Warren et al. determined an efficient means of reprogramming multiple human cell types using modified mRNA that can express the four primary reprogramming proteins. These cells are referred to as induced pluripotency stem cells (iPSCs). Warren et al. found that enzymatically synthesized RNA substituted with Pseudo-UTP, 5-Methyl-CTP and ARCA effectively evaded the cell’s innate immune response, a crucial component in their success. Reduced toxicity due to substitution with Pseudo-UTP, 5-Methyl-CTP was critical since it allowed repeated transfection with in vitro transcribed mRNA over several weeks.

https://www.targetmol.com/compound/mrtx-1719

[[File:Pseudouridine-5'-Triphosphate.png|400px|RTENOTITLE]]

5-Methyluridine-5'-Triphosphate ,&nbsp; 5-Methyl-UTP, 5-Me-UTP, 5-mUTP, Trilink:&nbsp;N-1024-5,5 µmole $464.

Thermostability Enhancer

[[File:5-Methyluridine-5'-Triphosphate.png|RTENOTITLE]]

N1-Methyl-Pseudouridine-5'-Triphosphate,&nbsp;N1-Methylpseudo-UTP, 1-Methylpseudo-UTP, Trilink: N-1081-5,5 µmole $464

Incorporation of N1-methylpseudouridine can reduce the immunogenicity of the resulting mRNA

[[File:N1-Methyl-Pseudouridine-5'-Triphosphate.png|RTENOTITLE]]

https://www.targetmol.com/compound/uridine_5_triphosphate_tetrasodium

'''Reagents not supplied:'''

RiboMAX™ Large Scale RNA Production Systems – T7: Promega P1300<br/>ScriptCap™ m7G Capping System: CellScript C-SCCE0625<br/>ScriptCap™ 2'-O-Methyltransferase Kit: CellScript C-SCMT0625<br/>A-Plus™ Poly(A) Polymerase Tailing Kit: CellScript C-PAP5104H<br/>Antarctic Phosphatase: New England BioLabs M0289S<br/>ScriptGuard™ RNase Inhibitor: CellScript C-SRI6310K

Lipofectamine™ MessengerMAX™ Transfection Reagent: ThermoFisher LMRNA001<br/>RiboJuice™ mRNA Transfection Kit: MilliporeSigma TR-1013

== Design of Transgene into T7-VEE ==

1, add Kozak after Nde1

Nde1&nbsp; &nbsp; &nbsp;Kozak&nbsp; &nbsp; &nbsp;start

catatg&nbsp;gaa t tcc gcc acc ATG---

2, Add stop codon and Kozak after Nde1

Nde1&nbsp; &nbsp; &nbsp; &nbsp; stop&nbsp; &nbsp; &nbsp;Kozak start

catatg agg ttc tag gcc acc ATG
<pre>                          -17                   +1-->
T7 promoter class III 5'- TAA TAC GAC TCA CTA TA G GGA GAC ATG CTA - 3'
T7 promoter class II  5'- TAA TAC GAC TCA CTA TT A GGG AGA ATG CTA - 3'

T7 RNA Polymerase
+ rNTPs
--->  RNA 5'- pppGGGAGACAUGCUA

</pre>



== 5'UTR ==

1,&nbsp; T7 + beta globin + kozak

TAATACGACTCACTATAaGG '''GAATACAAGCTACTTGTTCTTTTTGCA '''AATTGCCACCATG

2, T7 +&nbsp;&nbsp;Venezuelan equine encephalitis virus&nbsp;

TAATACGACTCACTATAaGG '''ATGGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAA'''

== Template preparation ==

1. Double-stranded DNA template

'''**NOTE: CleanCap AG requires modification to the sequence just downstream of the T7 promoter sequence, replacing the “GG” that follows the T7 promoter sequence with an “AG” as follows:'''





'''Standard T7 promoter (underlined) with&nbsp;''''''GG'''''':'''

'''5’&nbsp;TAATACGACTCACTATA''''''GG''''''&nbsp;3’'''

'''Sequence required for CleanCap<sup>®</sup>&nbsp;with AG:'''

'''5’&nbsp;TAATACGACTCACTATA''''''AG''''''&nbsp;3’'''

'''New primer'''

'''T7-mRNA-F&nbsp;'''<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">&nbsp;(for pCDNA5 plasmid) (Box&nbsp;</span>O1<span style="white-space: pre;">well </span>I3)

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">AAAT'''TAATACGACTCACTATA'''''AG''</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''T7-mRNA-F2''' (for pCDNA5 pCMV5-frt plasmid)</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">AT'''TAATACGACTCACTATA'''''AG''GAGACCCAAG</span>



<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''T7-mRNA-F3'''&nbsp;(for plasmid with traditional T7 promoter and kozak seq, such as pMiniT2--IRES)</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''TAATACGACTCACTATA'''''AG''GAGG</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''T7-mRNA-F4''' (for plasmid vector pCAGGS, </span>2724<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">)</span>

'''TAATACGACTCACTATA'''''AGGCAAGGCCACCGCCACCATG''

Reverse primer for 2724&nbsp;pCAGGS:&nbsp;pCAGGS-mRNA-R2

tTTTTTTTTTTTTTTTTTTTTTTTAAATGATTTGCCCTCC<br/>(GGAGGGCAAATCATTTAAAAAAAAAAAAAAAAAAAAAAAa)

reverse primer for C-Flag tag

'''Flag-R'''<br/>(CAAAGACGATGACGACAAGTAACTCGAGAAAAAAAAAAAAAAA)<br/>&nbsp;TTTTTTTTTTTTTTTCTCGAGTTACTTGTCGTCATCGTCTTTG





<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">2. NEB Monarch<sup>®</sup>&nbsp;RNA Cleanup Kit (500 µg),&nbsp; &nbsp;[https://www.neb.com/products/t2050-monarch-rna-cleanup-kit-500-ug https://www.neb.com/products/t2050-monarch-rna-cleanup-kit-500-ug]&nbsp;T2050&nbsp;</span>

'''For pMiniT2-pA vector&nbsp;''' pA-T7-F<br/>TAATACGACTCACTATAGGGAGAC

pA-mRNA-R<br/>TAGGTAATCGTCACCTGcag<br/>(ctgCAGGTGACGATTACCTA)

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''Co-transcriptional Capping Protocol:'''<br/>('''NOTE''': <span style="color:#FF0000;">'''5 mM final each NTP'''</span> instead of 10 mM in standard HiScribe Reaction)</span>

#<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Thaw the necessary components, keep the T7 RNA Polymerase Mix on ice.&nbsp;</span>
#<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Mix and pulse-spin in a microfuge to collect the solutions to the bottom of the tubes.&nbsp;</span>
#<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Set up the reaction at&nbsp;'''room temperature'''&nbsp;in the following order:</span>

{| border="1" cellspacing="0" cellpadding="0"
|-
! width="259" | 
<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''Component'''</span>

! width="134" | 
<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''Volume'''</span>

! width="197" | 
<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''Concentration'''</span>

|-
| 
<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''Nuclease-free Water'''</span>

| 
<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''X µl'''</span>

| 


|-
| 
<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''10X Reaction Buffer'''</span>

| 
<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''2 µl'''</span>

| 
<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''0.5X final'''</span>

|-
| 
<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''100 mM ATP'''</span>

| 
<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''2 µl'''</span>

| 
<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''5 mM final'''</span>

|-
| 
<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''100 mM GTP'''</span>

| 
<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''2 µl'''</span>

| 
<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''5 mM final'''</span>

|-
| 
<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''100 mM pseudo-UTP'''</span>

| 
<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''2 µl'''</span>

| 
<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''5 mM final'''</span>

|-
| 
<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''100 mM 5m-CTP&nbsp;'''</span>

| 
<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''2 µl'''</span>

| 
<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''5 mM final'''</span>

|-
| Cyanine 5-UTP, optional<br/>
| 0.5 '''µl'''
| <br/>
|-
| 
<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''100 mM CleanCap''''''<sup>®</sup>''''''&nbsp;AG or AG (3’ OMe) (N-7113 or N-7413)'''</span>

| 
<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''<span style="color:#FF0000;">1.6</span> µl'''</span>

| 
<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''4 mM final'''</span>

|-
| 
<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''Linear Template DNA'''</span>

| 
<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''X µl'''</span>

| 
<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''<span style="color:#FF0000;">1 µg</span> total'''</span>

|-
| 
<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''T7 RNA Polymerase Mix'''</span>

| 
<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''<span style="color:#FF0000;">2 </span>µl'''</span>

| 


|-
| 
<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''Total'''</span>

| 
<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''<span style="color:#FF0000;">40</span> µl'''</span>

| 


|}

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">1.Mix thoroughly by pipet and pulse-spin in a microfuge. Incubate at 37°C for 2 hours in a dry air incubator or PCR machine to prevent evaporation.</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">2. Follow the protocol provided with the HiScribe T7 High Yield RNA Synthesis Kit (NEB E2040S ) for DNase treatment and RNA purification.</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">3. Add TURBO DNase (ThermoFisher Scientific, incubated at 37 ◦C for 30 min Purify with column</span>

4, Add 5 µL of 10×Phosphatase Buffer to the reaction.&nbsp;

5, Add 1 µL of RNase-free DNase&nbsp;&nbsp;



<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">6,&nbsp; Add 2&nbsp;</span>µL&nbsp;<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Antarctic Phosphatase (New England Biolabs) inccubate 37 ◦C for 30 min (&nbsp;Phosphatase treatment</span>&nbsp;reduces innate immune response in mammalian cells)



<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'</span>''5m-CTP''''''&nbsp;''': 5-Methylcytidine-5'-Triphosphate

'''pseudo-UTP'''''':&nbsp;'''Pseudouridine-5'-Triphosphate

Cyanine 5-UTP, 100 nmol&nbsp;[https://www.perkinelmer.com/product/cyanine-5-utp-nel583001ea NEL583001EA $646.00&nbsp;]&nbsp; ,&nbsp;[https://www.enzolifesciences.com/ENZ-42506/cyanine-5-utp-enhanced/ ENZ-42506, 250 nmol, $354.00&nbsp;]&nbsp; ,&nbsp;[https://www.scbt.com/p/cyanine-5-utp sc-499469, 250 nmol $340]

Cyanine 3-UTP, 100 nmol&nbsp;[https://www.perkinelmer.com/product/cyanine-3-utp-nel582001ea NEL582001EA&nbsp; $641]&nbsp;[https://www.apexbt.com/cy3-utp.html 25ul (10mM) $195&nbsp;]&nbsp;&nbsp;[https://www.scbt.com/p/cyanine-3-utp sc-499471 250 nmol $350]

== <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Large scale production</span>'''</span> ==

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">CleanCap AG (TriLink N-7113) and&nbsp;RiboMAX™ Large Scale RNA Production Systems-T7 (Promega P1300)</span>

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Component</span><br/>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Large Scale</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Mid Scale</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Small scale</span>
|-
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">T7 Transcription Buffer 5X</span><br/>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">20 μL</span><br/>
| <br/>
| <br/>
|-
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">100 mM ATP</span><br/>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">7.5&nbsp;μL</span>
| <br/>
| <br/>
|-
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">100 mM GTP</span><br/>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">7.5&nbsp;μL</span><br/>
| <br/>
| <br/>
|-
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">100 mM 5mCTP</span><br/>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">7.5&nbsp;μL</span><br/>
| <br/>
| <br/>
|-
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">100 mM pseudo-UTP</span><br/>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">7.5&nbsp;μL</span><br/>
| <br/>
| <br/>
|-
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">CleanCap AG, 100 mM&nbsp;</span><br/>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">7.5&nbsp;μL</span><br/>
| <br/>
| <br/>
|-
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">DNA plasmid (1 μg/μL)</span><br/>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">10 μL</span><br/>
| <br/>
| <br/>
|-
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">RNAse-free water</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">22.5&nbsp;&nbsp;μL</span>
| <br/>
| <br/>
|-
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Enzyme Mix (T7)</span><br/>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">10 μL</span><br/>
| <br/>
| <br/>
|-
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Total</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">100 μL</span><br/>
| <br/>
| <br/>
|-
| colspan="4" | <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Incubate at 37°C for 2 hrs.&nbsp;Do not incubate beyond 2 hours at 37°C as a longer incubation period may result in increased RNA degradation.</span><br/>
|-
| <br/>
| <br/>
| <br/>
| <br/>
|-
| colspan="4" | <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Add 10 μL RQ1 RNase-free DNase (Promega M6101) for 10 μg of template DNA (use 1 μL per μg DNA template). Gently mix by pipetting up and down. Incubate for 15 minutes at 37˚C.</span><br/>
|-
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Yield</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">300-500 μg.</span><br/>
| <br/>
| <br/>
|}



<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">For making PCR fragment for in vitro transcription</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''Method 1:''' making T7 promoter + BGH tail</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">CMV-seq-F&nbsp;</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">GAGGTCTATATAAGCAGAGCTC</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">BGH-mRNA-R</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">GGTTCTTTCCGCCTCAGAAG</span>



<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''Method 2''': making modified promoter AG + BGH tail</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''T7-mRNA-F1&nbsp;'''<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">&nbsp;(for pCDNA5 plasmid)&nbsp;</span></span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;"><span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">AAAT'''TAATACGACTCACTATA'''''AG''</span></span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;"><span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''or T7-mRNA-F2'''&nbsp;(for GOI in pCDNA5 and pCMV5-Frt plasmid vector)</span></span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;"><span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">AT'''TAATACGACTCACTATA'''''AG''GAGACCCAAG</span></span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">BGH-mRNA-R&nbsp;</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">GGTTCTTTCCGCCTCAGAAG&nbsp;</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">with Flag Tag and polyA: use&nbsp;'''<span style="color: rgb(0, 0, 0); font-family: Arial, Verdana, sans-serif; font-size: 12px; white-space: nowrap; background-color: rgb(251, 251, 239);">Flag-R</span>'''</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">&nbsp;TTTTTTTTTTTTTTTCTCGAGTTACTTGTCGTCATCGTCTTTG</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">(CAAAGACGATGACGACAAGTAACTCGAGAAAAAAAAAAAAAAA)<br/>PCR product sequence</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;"><span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">AT</span>'''TAATACGACTCACTATA'''''AG''<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">GAGACCCAAG</span></span>
<pre style="color: rgb(0, 0, 0); font-size: 12px; background-color: rgb(251, 251, 239);"><span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">ATTAATACGACTCACTATAAGGAGACCCAAGCTGGCTAGC
GCTAGC CACCATG--GOI --ggatcc gactaCAAAGACGATGACGACAAGTAACTCGAGAAAAAAAAAAAAAAA</span></pre>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''Method 3:''' make T7 promoter + IRES+ BGH tail</span>



<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;"><u>first PCR</u></span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">fragment 1</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">&nbsp;2711-IRES-F</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">ACGATGATAATATGGCCAGCTAGCCACCATGTTCGTCTTC&nbsp;</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">BGH-mRNA-R&nbsp;</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">GGTTCTTTCCGCCTCAGAAG&nbsp;</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">fragment 2</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">T7-mRNA-F3 (for plasmid with traditional T7 promoter and kozak seq, such as pMiniT2--IRES )</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">TAATACGACTCACTATAAGGAGG</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">IVT-IRES-R</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">CATGGTGGTGGCCATATTATC</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">2nd PCR</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;"><span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''T7-mRNA-F3'''&nbsp;(for plasmid with traditional T7 promoter and kozak seq, such as pMiniT2--IRES)</span></span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;"><span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''TAATACGACTCACTATA'''''AG''GAGG</span></span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">TAATACGACTCACTATAAGGAGgccACCATG</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">GH-mRNA-R&nbsp;</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">GGTTCTTTCCGCCTCAGAAG</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''For GOI in pCMV5-Frt vector&nbsp;''' '''T7-mRNA-F3'''<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">&nbsp;&nbsp;</span></span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''TAATACGACTCACTATA'''''AG''<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">GAGG</span></span>



<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;"><span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''Method 4:'''&nbsp;make T7 promoter + polyA</span></span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;"><span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">For example: 2683 pJet1.2-SARS2-S1-1 PCR using</span></span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;"><span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">T7-mRNA-F3 '''TAATACGACTCACTATAAGGAGG'''</span></span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;"><span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">on fragment on pJet1.2</span></span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;"><span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''TAATACGACTCACTATAAGGAGG''' TAATACGACTCACTATAGGGAGgccAccATGAGAACTCAATTACCCCCTGCATACACTAATTCTTTCACACGTGGTGTTTATTACCCTGACAAAGTTTTCAGATCCTCAGTTTTACATTCAACTCAGGACTTGTTCTTACCTTTCTTTTCCAATGTTACTTGGTTCCATGCTATACATGTCTCTGGGACCAATGGTACTAAGAGGTTTGATAACCCTGTCCTACCATTTAATGATGGTGTTTATTTTGCTTCCACTGAGAAGTCTAACATAATAAGAGGCTGGATTTTTGGTACTACTTTAGATTCGAAGACCCAGTCCCTACTTATTGTTAATAACGCTACTAATGTTGTTATTAAAGTCTGTGAATTTCAATTTTGTAATGATCCATTTTTGGGTGTTTATTACCACAAAAACAACAAAAGTTGGATGGAAAGTGAGTTCAGAGTTTATTCTAGTGCGAATAATTGCACTTTTGAATATGTCTCTCAGCCTTTTCTTATGGACCTTGAAGGAAAACAGGGTAATTTCAAAAATCTTAGGGAATTTGTGTTTAAGAATATTGATGGTTATTTTAAAATATATTCTAAGCACACGCCTATTAATTTAGTGCGTGATCTCCCTCAGGGTTTTTCGGCTTTAGAACCATTGGTAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACTTTACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTT</span></span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;"><span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">CTTCAGGTTGGACAGCTGGTTAGTAACCGCTGAGCAATAACTAGCAAAAAAAAAA PCR Template Preparation<br/>1. Cloning of IDT gene fragment into pMiniT2 or pJet1.2<br/>2683 (DS1)<br/>TAATACGACTCACTATAGGGAGgccAccATGAGAACTCAATTACCCCCTGCATACACTAATTCTTTCACACGTGGTGTTTATTACCCTGACAAAGTTTTCAGATCCTCAGTTTTACATTCAACTCAGGACTTGTTCTTACCTTTCTTTTCCAATGTTACTTGGTTCCATGCTATACATGTCTCTGGGACCAATGGTACTAAGAGGTTTGATAACCCTGTCCTACCATTTAATGATGGTGTTTATTTTGCTTCCACTGAGAAGTCTAACATAATAAGAGGCTGGATTTTTGGTACTACTTTAGATTCGAAGACCCAGTCCCTACTTATTGTTAATAACGCTACTAATGTTGTTATTAAAGTCTGTGAATTTCAATTTTGTAATGATCCATTTTTGGGTGTTTATTACCACAAAAACAACAAAAGTTGGATGGAAAGTGAGTTCAGAGTTTATTCTAGTGCGAATAATTGCACTTTTGAATATGTCTCTCAGCCTTTTCTTATGGACCTTGAAGGAAAACAGGGTAATTTCAAAAATCTTAGGGAATTTGTGTTTAAGAATATTGATGGTTATTTTAAAATATATTCTAAGCACACGCCTATTAATTTAGTGCGTGATCTCCCTCAGGGTTTTTCGGCTTTAGAACCATTGGTAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACTTTACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTTCTTCAGGTTGGACAGCTGGTTAGTAACCGCTGAGCAATAACTAGCAAAAAAAAAA</span></span>

<br/><span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;"><span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">2. Make miniprep, send miniprep DNA out for sequencing (Genewiz, Macrogen)<br/>3, PCR&nbsp;&nbsp;<br/>1 ul of plasmid (50~100 ng)<br/>1 ul of 10 uM T7-mRNA-F3 TAATACGACTCACTATAAGGAGG<br/>1 ul 1 uM 2683-R2 2683-R2 TTACTTGTCGTCATCGTCTTTGTAGTCACCAGCTGTCCAACCTGAAG (CTTCAGGTTGGACAGCTGGTGACTACAAAGACGATGACGACAAGTAA)<br/>1 ul 10 uM Flag-R&nbsp;<br/>TTTTTTTTTTTTTTTCTCGAGTTACTTGTCGTCATCGTCTTTG</span></span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;"><span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">(CAAAGACGATGACGACAAGTAACTCGAGAAAAAAAAAAAAAAA)<br/>21 ul H2O<br/>+ 25 ul 2 X Phusion PCR mix (NEB)</span></span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;"><span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">PCR:<br/>2 min at 95 oC<br/>30 cycles of 95 oC 30 sec, 55 oC 30 sec, 72 oC 1 min<br/>7 min at 72 oC.<br/>Final Product 802 bp<br/>TAATACGACTCACTATAAGGAGG<br/>ccAccATGAGAACTCAATTACCCCCTGCATACACTAATTCTTTCACACGTGGTGTTTATTACCCTGACAAAGTTTTCAGATCCTCAGTTTTACATTCAACTCAGGACTTGTTCTTACCTTTCTTTTCCAATGTTACTTGGTTCCATGCTATACATGTCTCTGGGACCAATGGTACTAAGAGGTTTGATAACCCTGTCCTACCATTTAATGATGGTGTTTATTTTGCTTCCACTGAGAAGTCTAACATAATAAGAGGCTGGATTTTTGGTACTACTTTAGATTCGAAGACCCAGTCCCTACTTATTGTTAATAACGCTACTAATGTTGTTATTAAAGTCTGTGAATTTCAATTTTGTAATGATCCATTTTTGGGTGTTTATTACCACAAAAACAACAAAAGTTGGATGGAAAGTGAGTTCAGAGTTTATTCTAGTGCGAATAATTGCACTTTTGAATATGTCTCTCAGCCTTTTCTTATGGACCTTGAAGGAAAACAGGGTAATTTCAAAAATCTTAGGGAATTTGTGTTTAAGAATATTGATGGTTATTTTAAAATATATTCTAAGCACACGCCTATTAATTTAGTGCGTGATCTCCCTCAGGGTTTTTCGGCTTTAGAACCATTGGTAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACTTTACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTT<br/>CTTCAGGTTGGACAGCTGGTGACTACAAAGACGATGACGACAAGTAACTCGAGAAAAAAAAAAAAAAA</span></span>

<br/><span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;"><span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">MRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGDYKDDDDK</span></span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;"><span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">purify PCR fragment using Qiagen gel purification kit</span></span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;"><span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Use 1 ug for IVT <font color="#252525"><font face="sans-serif"><font size="3" style="font-size: 12pt">Purify PCR fragment using Qiagen gel purification kit</font></font></font></span></span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;"><span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;"><font color="#252525"><font face="sans-serif"><font size="3" style="font-size: 12pt">Use 1 ug for IVT</font></font></font></span></span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;"><span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">[[File:2683 PCR for mRNA.JPG|RTENOTITLE]]</span></span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;"><span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">purify PCR fragment</span></span>



<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;"><span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">use 1 ug for IVT</span></span>

== <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Add an EGFP tag to Flag tag Clone</span> ==

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''EGFP PCR template&nbsp;'''</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">T7-EGFP-F<br/>TAATACGACTCACTATAAGGAGACCATGGTGAGCAAGGGC</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">EGFP-R<br/>tttttttTTTTTTTTTTTTTTTTACTTGTACAGCTCGTCC<br/>(GGACGAGCTGTACAAGTAAAAAAAAAAAAAAAAaaaaaaa)</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''SARS2-EGFP fusion PCR template'''</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">clones should already have a Flag tag</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">to PCR out clone, such as 2686, 2689, 2690, 2691 original IDT fragments&nbsp;in pJet1.2</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''T7-mRNA-F4'''</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">TAATACGACTCACTATAAGGAGgccACCATG</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Flag-EGFP-R<br/>ctcctcgcccttgctcaccttgtcgtcatcgtctttgtag<br/>and PCR EGFP from&nbsp;2743 pLVX-Spike-IRES-EGFP</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Flag-EGFP-F<br/>ctacaaagacgatgacgacaaggtgagcaagggcgaggag</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">EGFP-R<br/>tttttttTTTTTTTTTTTTTTTTACTTGTACAGCTCGTCC<br/>(GGACGAGCTGTACAAGTAAAAAAAAAAAAAAAAaaaaaaa)</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">gel purified two fragments and combine</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">PCR again using&nbsp;</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''T7-mRNA-F4'''</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">EGFP-R<br/>tttttttTTTTTTTTTTTTTTTTACTTGTACAGCTCGTCC<br/>(GGACGAGCTGTACAAGTAAAAAAAAAAAAAAAAaaaaaaa)</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">purify and use it as template for IVT</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">transfection 293T cells with&nbsp;purified mRNA&nbsp; to see GFP expression</span>



== <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">For amplifying DNA template from pCDNA5-FRT</span> ==

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">T7-mRNA-F2</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;"><span style="font-weight:600;">ATTAATACGACTCACTATA</span><span style="font-style:italic;">AG</span><span style="font-weight:600;">GAGACCCAAG</span></span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">pCDNA5-mRNA-R<br/>TTTTTTTTTTTTTTTTTTTTGGGGTCGAGGTGCCGTAAAG<br/>(CTTTACGGCACCTCGACCCCAAAAAAAAAAAAAAAAAAAA)</span>

== <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Current Working Protocol&nbsp;</span> ==

=== <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">mRNA for transfection in vitro</span> ===

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Template preparation by PCR using 2X Taq PCR mix</span>

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Clone #</span><br/>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">F Primer</span><br/>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">R Prime</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">PCR Length</span>
|-
| <br/>
| <br/>
| <br/>
| <br/>
|-
| <br/>
| <br/>
| <br/>
| <br/>
|-
| <br/>
| <br/>
| <br/>
| <br/>
|-
| <br/>
| <br/>
| <br/>
| <br/>
|}

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">After PCR, take&nbsp;5 ul to check purity on agarose gel.&nbsp;&nbsp;Purify the remaining&nbsp;PCR product using a spin column, elute DNA using 25 ul ddH2O, measure DNA concentration using nanodrop</span>

=== <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">mRNA as vaccine for&nbsp;animal study</span> ===

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Template preparation by PCR using 2X Taq PCR mix</span>

{| border="1" cellspacing="1" cellpadding="1" style="width: 577px;"
|-
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Clone #</span><br/>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Name</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Des</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">F Primer</span><br/>
| style="width: 165px;" | <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">R Prime</span>
| style="width: 37px;" | <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">PCR Length</span>
|-
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">2684</span><br/>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">DS2</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">T7-IRES-DS2</span>
| 
<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">T7-IRES-F</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">or T7-F</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">or&nbsp;IVT-T7-F</span>

| style="width: 165px;" | <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Flag-R</span><br/>
| style="width: 37px;" | <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">997</span>
|-
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">2838</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">DS2</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">T7-DS2 in pJet</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">T7-DS2-F</span><br/>
| style="width: 165px;" | <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Flag-R</span>
| style="width: 37px;" | <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">454</span>
|-
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">2831</span><br/>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">DS2-fd</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">T7-DS2-fd&nbsp;in pJet</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">T7-DS2-F</span><br/>
| style="width: 165px;" | <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Foldon-R</span><br/>
| style="width: 37px;" | <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">500</span>
|-
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">2823</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">DS2</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">T7-DS2&nbsp;in pJet</span><br/>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">T7-DS2-F</span><br/>
| style="width: 165px;" | <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Flag-R</span><br/>
| style="width: 37px;" | <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">454</span><br/>
|-
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">2823</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">DS2&nbsp;<span style="color: rgb(0, 0, 0); font-family: Arial, Verdana, sans-serif; font-size: 12px;">SAM</span></span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">T7-DS2&nbsp;in pT7-VEE</span><br/>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">T7 or T7-VEE-F</span>
| style="width: 165px;" | <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Flag-R or&nbsp;VEE-pA-R</span><br/>
| style="width: 37px;" | <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">8025</span>
|-
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">2831</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">DS2-fd SAM</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">T7-DS2-fd in pT7-VEE</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">T7&nbsp;or T7-VEE-F</span>
| style="width: 165px;" | <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Foldon-R&nbsp;or&nbsp;VEE-pA-R</span><br/>
| style="width: 37px;" | <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">8065</span>
|-
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">2874</span><br/>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">RBD-fd</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Full length RBD fuse to fd, with polyA</span>
| <br/>
| style="width: 165px;" | <br/>
| style="width: 37px;" | <br/>
|}

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Flag-R TTTTTTTTTTTTTTTCTCGAGTTACTTGTCGTCATCGTCTTTG</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">(CAAAGACGATGACGACAAGTAACTCGAGAAAAAAAAAAAAAAA)</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">T7-DS2-F TAATACGACTCACTATAAGGAGGCCACCATGGTCAGACAAATC</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Foldon-R&nbsp;tttttttttttttttttttttacaagaaggtcgacagcag</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">(ctgctgtcgaccttcttgtaaaaaaaaaaaaaaaaaaaaa)</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">VEE-pA-R&nbsp;&nbsp;GAATTAATTCCCCTCGAcgcgttctag</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">T7-VEE-F T<u>AATACGACTCACTATA</u>''<span style="color:#FF0000;">'''A'''</span>G''ATGGGCGGCGCATGAGAG</span>



<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">After PCR, take&nbsp;5 ul to check purity on agarose gel.&nbsp;&nbsp;Purify the remaining&nbsp;PCR product using a spin column, elute DNA using 25 ul ddH2O, measure DNA concentration using nanodrop</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">[[File:DS2 DS2fd PCR.JPG|RTENOTITLE]]</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">[[File:2838 2831 PCR1.JPG|RTENOTITLE]]</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">[[File:2838 2831 PCR2.JPG|RTENOTITLE]]</span>

== <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Use&nbsp;EMCV IRES to replace cap</span> ==

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">PCR EMCV IRES from 959,961,960</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">T7 promoter:&nbsp;TAATACGACTCACTATAGGG</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">EMCV-IRES-R GGTGGTGGCCATATTATCATC</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">PCR from GOI&nbsp;2684&nbsp;pJet1.2-SARS2-S1-2</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Spike RDB DS2</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">F: GATGATAATATGGCCACCACCCATATGGTCAGACAAATCGCTCCAG</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">R:&nbsp;</span>

== <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Make Self-amplifying mRNA (SAM) using VEE vector</span> ==

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">PCR amplify</span>



== <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">PCR making template from pCAGGS vector</span> ==

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">for making mRNA PCR using<br/>pCAGGS-T7<br/>TAATACGACTCACTATAAGcattttggcaaggccaccatg<br/>pCAGGS-pA<br/>tTTTTTTTTTTTTTTTTTTTTTCATGGCAGCCAGCATATG<br/>(CATATGCTGGCTGCCATGAAAAAAAAAAAAAAAAAAAAAa)</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">template:&nbsp;2725&nbsp;pCAGGS-SARS2-RBD or&nbsp;2754&nbsp;pCAGGS-SARS2-S-6H</span>



== <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">mRNA from 2874&nbsp;pCDNA5-frt-SARS2-RBD-fd&nbsp;</span> ==

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">RBD trimer, signal peptide, Codon optimized, 190 poly A tail</span>

=== <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">1, PCR</span> ===

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">primers</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">T7-mRNA-F2 ATTAATACGACTCACTATAAGG AGACCCAAG</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">2874-R<br/>AACGGGCCCTCTAGActcgAGAATTAATTCCCCTCGAcgc<br/>(gcgTCGAGGGGAATTAATTCTcgagTCTAGAGGGCCCGTT)</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">extension time 1 min</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">precipitate using isopropanol</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">optionally, remove extra nt from 3' using PacI or Xho1&nbsp;</span>

=== <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">2, Digestion plasmid DNA with <s>PacI, or Xho1 or</s> MluI or XbaI</span> ===

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">PacI digested template has many large RNA or DNA bands,</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">For T7-VEE, PacI&nbsp; and XhoI produces 3´overhang, so they are not good for linearization, if PacI or Xho1 is to be used, DNA template must be blunted by&nbsp; adding DNA Polymerase I Large (Klenow) Fragment at a concentration of 5u/μg and incubate the reaction&nbsp;mixture for 15 minutes at 22°C.&nbsp;&nbsp;</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''For &nbsp;digestion of 36 ug of T7-VEE plasmid in 200&nbsp;μL'''</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">36 μL T7-VEE Plasmid (1 μg/μL)</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">20 μL CutSmart Buffer (10X)</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">139 μL Sterile distilled water</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">5 μL MluI-HF (10,000 units/mL, NEB)</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Incubate digestion mixtures at 37°C for 3 hours.</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Add the same volume (200 μL) of phenol/chloroform/isoamyl alcohol (25:24:1) to each eppendorf tube containing the digestion mixture.<br/>Vortex the eppendorf tubes for 60 seconds. Centrifuge at high speed (14,000 rpm) for 5 minutes.<br/>Transfer each aqueous phase (top layer) into new eppendorf tubes.</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Note: T7-VEE plasmid is a large sized DNA and may be trapped in the interphase layer with the proteins. When the interphase is sticky (DNA is trapped), collect the interphase layer along with the aqueous layer.<br/>To remove the excess phenol, add an equal volume (200 μL) of chloroform/isoamyl alcohol (24:1) to each eppendorf tubes.<br/>Vortex the eppendorf tubes for 60 seconds and centrifuge at high speed (14,000 rpm) for 5 minutes.<br/>Transfer each aqueous phase (top layer) into new eppendorf tubes.<br/>Add 1/10 volume (20 μL) of 3 M sodium acetate, pH 5.2 (this can be found in the RiboMAX Large Scale RNA Production kit) and 2.5 X volume (500 μL) of 100% ethanol to each eppendorf tubes.<br/>Vortex and place at -80°C for 5 minutes or store at -20°C until use.<br/>Note: If there are no plans to proceed to RNA synthesis on the same day, stop at this step. DNA will be stable for several months at -20°C.<br/>Centrifuge at high speed (14,000 rpm) for 10-15 minutes at 4°C to pellet the DNA plasmid. Remove the ethanol carefully with a pipet tip, being careful not to dislodge the DNA pellet.<br/>Gently add 1 mL 70% ethanol to wash the DNA pellet. Centrifuge at high speed at 4°C for 1 minute. Remove the ethanol carefully with a pipet tip, being careful not to dislodge the RNA pellet.<br/>Centrifuge shortly, and carefully remove any visible residual ethanol with a P20 pipette. Drying the DNA pellet is not necessary. In general, approximately 20% of plasmid may be lost during purification.<br/>Resuspend the DNA pellet in a small volume (15~20 μL, depending on starting amount) of DNase & RNase-free water before measuring the concentration.<br/>Measure the DNA concentration of each linearized DNA plasmid. Adjust the concentration to 1 μg/μL.</span>





<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''mRNA analysis by agarose'''</span>

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Lane</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Desc</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Note</span>
|-
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">1</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">PacI digested plasmid, large scale</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">need to remove DNA using DNase I</span>
|-
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">2</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">PacI digested plasmid,&nbsp;small scale</span><br/>
| <br/>
|-
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">3</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Xho1 digested plasmid,&nbsp;small scale</span>
| <br/>
|-
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">4</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">PCR template,&nbsp;small scale</span>
| <br/>
|-
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">5</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">DNA ladder, NEB, 1 Kb</span>
| <br/>
|-
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">6</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">PCR template 1, precipitate with isopropanol + glycogen</span><br/>
| <br/>
|-
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">7</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">PCR template 2, precipitate with isopropanol</span><br/>
| <br/>
|}

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">[[File:ShenDong mRNA QC 12 14 2020 long.JPG|RTENOTITLE]]</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">mRNA made from 6 + 7 combined, 4 tubes of 40 ul reaction from template 6,&nbsp;4 tubes of 40 ul reaction from template 7, purified using Qiagen RNeasy miniprep kit</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">[[File:MRNA2.jpg|RTENOTITLE]]</span>



<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">CleanCap is used for easy one -step reaction, while enzymatic capping method requires three-step reaction but it minimizes the IFN responses by combination with phosphatase treatment</span>

== <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">High yield Production of mRNA</span> ==

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">10 mL transcription reaction mixture<br/>30 mM Tris (pH 8.1 at 37°C), 15 mM Mg2+ , 10 mM dithiothreitol (DTT), 2 mM spermidine, 0.01% (v/v) Triton X-100, 4 mM each NTP, 1 mL of PCR-generated DNA template, and 0.1 mg/mL of T7 RNAP</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">optimal concentration of Mg2+ is 15 ~45 mM<br/>After 2.5 h of incubation at 37°C, pyrophosphate, which forms during in vitro transcription reaction, was pelleted down by centrifugation, and additional Mg2+ should be added to the reaction.&nbsp;<br/>The reaction continued till 5 h.&nbsp;<br/>Size-exclusion FPLC based RNA purification<br/>RNA sample preparation for chromatographic purification<br/>Transcription mixtures for the S-box RNA were heated to 90°C for 5 min, immediately transferred to ice for 5 min and then pelleted down (centrifuged at 16, 000g for 10 min). The supernatant was collected and mixed as 1: 4 with Tris-EDTA buffer containing 8 M urea and 100 mM NaCl before loading onto size-exclusion FPLC (SEC) columns. Sample pre-load treatment was operated to remove the T7 RNAP, residual pyrophosphates and aggregates formed during in vitro transcription.</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Denaturing chromatographic purification<br/>The glass columns (890/10 mm) and (xk16/1000 mm) were packed according to manufacturer’s specifications with gel filtration matrix (Superdex-75, Superdex-200) and equilibrated in a cold room (4°C) with several column volumes (CV) of specified buffer. The transcription mixture from the previous step was loaded directly onto the equilibrated SEC column, and the chromatography was performed at 1 mL/min flow rate, collecting 2 mL fractions. Fractions were analyzed by denaturing urea-PAGE. S-box RNA containing fractions were combined and concentrated to 0.5 mL using Millipore centrifugal filter units (EMD Millipore), washed twice with the appropriate buffer for chemical probing analysis. All experiments were performed at 4°C. The efficiency of proposed purification system was confirmed through SHAPE experiments for S-box RNA.</span>

https://www.targetmol.com/compound/mrtx-1719

== <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Transfection</span> ==

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Thaw mRNAs were thawed on ice and denatured at 65°C for 3min</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Incubation at room temperature for 45min.</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''1. Lipofectamine 3000'''</span>

*<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">On day 0, HEK 293T cells are seeded at a concentration of&nbsp;5 × 10<sup>5 </sup>cells/well in a 6-well plate to ascertain 70–90&nbsp;% of confluency on the day of transfection (day 1).</span>
*<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">For transfection, a ratio of 2 μl of Lipofectamine® reagent to 1 μg of RNA is used. Cells are transfected with 1 and 2 μg of&nbsp;mRNA. Transfection is performed in duplicates.</span>
*<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">On the day of transfection, an appropriate volume of Lipofectamine® reagent is prepared in Opti-MEM® medium and incubated at room temperature for 5 min.</span>
*<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">&nbsp;1 and 2 μg of mRNA are diluted in Opti-MEM® medium. If required, pre-dilute mRNA to a concentration of 0.5 μg/μl.</span>
*<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Mix Lipofectamine® reagent and Opti-MEM®-diluted mRNA&nbsp;gently in a total volume of 500 μl at a 1:1 ratio (v/v) and incubated&nbsp;at room temperature for 20 min.</span>
*<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Wash cells twice with 500 μl DMEM medium containing glutamine&nbsp;but without FBS. Add 2 ml of DMEM to the cells.</span>
*<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Add 500 μl of mRNA–lipid complexes to each well. Mix gently.&nbsp;</span>
*<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Incubate cells at 37 °C in a CO2-incubator for 4–6 h.</span>
*<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">After the incubation, replace the medium with 2 ml of fresh DMEM medium containing 10&nbsp;% FBS.</span>
*

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''2. TT3 nanoparticles'''</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">lipid:DOPE:cholesterol:DMG-PEG2000 at a molar ratio of 20:30:40:0.75 ([https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4869688/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4869688/])</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">3. Protamine&nbsp;as an immunostimulatory component</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Protamine. Pharmaceutical Protamine is available at two&nbsp;concentrations: 1000 (1 ml neutralizes 1000 IU of heparin) or&nbsp;5000 (1 ml neutralizes 5000 IU of heparin). To generate particles&nbsp;of 130 nm, we use Protamine Ipex 5000 from MEDA&nbsp;Pharmaceuticals (Protamine hydrochloride 5000 IU/ml). The Protamine is stored at 4 °C&nbsp;</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Dissolve the protamine in Ringer lactate solution.<br/>&nbsp;While stirring, slowly add the protamine-Ringer lactate solution&nbsp;half of the mRNA until you reach a weight ratio of&nbsp;mRNA and protamine of 2:1.<br/>&nbsp;Stir the solution for another 10 min to ensure the formation of&nbsp;stable complexes.<br/>&nbsp;Add the remaining half of free mRNA and stir briefly.<br/>&nbsp;Adjust the final concentration of the vaccine with Ringer lactate&nbsp;solution.</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Carry out all procedures at room temperature unless otherwise&nbsp;specified.</span>

<br/><span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">This step is performed under laminar flow using sterile&nbsp;equipment.</span>

*<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Prepare a 0.5 mg/ml Protamine solution (see Note 5) by diluting&nbsp;10 μl of Protamine 5000 in 280 μl of water.&nbsp;</span>
*<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Prepare a 0.5 mg/ml RNA solution (see Note 5) by diluting&nbsp;the RNA stock with the appropriate amount of water.</span>
*<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Add an equal amount of Protamine solution to the RNA solution&nbsp;(see Note 6), and pipet quickly up and down at least ten&nbsp;times (see Note 7).</span>
*<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Leave the solution at room temperature for 10 min (see Note 8).&nbsp;1. Dilute 40 μl of the Protamine–RNA formulation (containing&nbsp;10 μg of RNA and 10 μg of Protamine) with 1 ml of water (see&nbsp;Note 9).</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Set the viscosity of the analyzer to 0.89 (select within the software&nbsp;setting either measurement in water or a viscosity of&nbsp;0.89). As shown in Fig. 1, particles made in the conditions&nbsp;described above are approximately 130 nm in size with a polydispersity&nbsp;index (PDI) of less than 0.3 (see Note 10).</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Protamine sulfate, such as Valeant Protamine, gave identical results.<br/>Freezing Protamine solutions may jeopardize&nbsp;its functionality as far as production of immunostimulating&nbsp;RNA polyplex is concerned. Pharmaceutical Protamine should&nbsp;be stored at 4 °C as indicated on the package.<br/>Any RNA (oligonucleotide, long uncapped and/or non-polyadenylatedmRNA, or capped mRNA) can be used to generate&nbsp;the 130 nm particles</span>

== <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Nonobody mRNA</span> ==

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">HiScribe T7 ARCA mRNA kit (with tailing; NEB; #E2060S). PCR product should be purified and run on an agarose gel to confirm amplicon size prior to its use as a template in the T7 ARCA mRNA transcription reaction. Generally, 0.1–0.5 mg of PCR fragments can be used in a 20-mL in vitro transcription reaction. After the preparation of PCR templates, standard mRNA synthesis with modified nucleotides should be started with thawing the necessary kit components, mixing, and pulse spinning in a microfuge to collect solutions to the bottoms of the tubes. The reaction was assembled at room temperature in the following order: nuclease-free water (to 20 uL), 2 x ARCA/NTP mix (10 uL), 5mCTP (10 mM, 2.5 uL), pseudo-UTP (10 mM, 2.5 uL), template DNA (1 mg), and T7 RNA polymerase mix (2 uL); mixed thoroughly; and pulse spun in a microfuge. Incubation was at 37oC for 30 min. DNase treatment to remove template DNA included adding 2 uL of DNase I, mixing well, and incubating at 37 oC for 15 min. poly(A) tailing starts with setting up the tailing reaction as below. Standard tailing reaction volume was 50 uL: H2O (20 uL), IVT reaction (20 uL), 10 X poly(A) polymerase reaction buffer (5 uL), and poly(A) polymerase. It was mixed thoroughly, pulse spun in a microfuge, and incubated at 37oC for 30 min. Synthesized mRNA can be purified by LiCl precipitation, phenol/chloroform extraction, followed by ethanol precipitation. To the 50-mL tailing reaction, 25 mL LiCl solution was added and mixed well. Incubation at -20 oC for 30 min was completed, as was centrifugation at 4oC for 15 min at top speed to pellet the RNA. The supernatant was removed carefully, the pellet was rinsed by adding 500 mL of cold 70% ethanol and centrifuged at 4oC for 10 min, ethanol was removed carefully, the tube was spun briefly to bring down any liquid on the wall, residual liquid was removed carefully using a sharp tip, the pellet was air dried, the mRNA was resuspend in 50 mL of 0.1 mM EDTA or a suitable RNA storage solution, and the RNA was heated at 65oC for 5– 10 min to completely dissolve the RNA and mixed well.</span>

== <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">LNP</span> ==

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">LNP were prepared by mixing ethanol and aqueous phase at a 1:3 volumetric ratio in a microfluidic device, using syringe pumps as previously described (Chen, Love et al., 2012). The ethanol phase was prepared by solubilizing a mixture of ionizable lipidoid cKK-E12, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, Avanti), cholesterol (Avanti), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(polyethyleneglycol)-2000] (ammonium salt) (C14-PEG 2000, Avanti) at a molar ratio of 35:16:46.5:2.5 The aqueous phase was prepared in 10 mM citrate buffer (pH 3) with either trastuzumab m 164 RNA or luciferase mRNA (Firefly luciferase mRNA, TranslateBio). LNPs were dialyzed against PBS in a Slide-A-Lyzer™ G2 Dialysis Cassettes, 20,000 MWCO (Thermo Fisher) for 2h at RT. The concentration of mRNA encapsulated into LNPs nanoparticles was analyzed using Quant-iT RiboGreen assay (Thermo Fisher), according to manufacturer’s protocol. The efficacy of mRNA encapsulation into LNPs was calculated by comparing measurements in the absence and presence of 1% (v/v) Triton X- 100. Nanoparticle size, polydispersity (PDI), and ζ-potential were analyzed by dynamic light scattering (DLS) using Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). LNP hydrodynamic diameters are reported in the volume weighting mode and are an average of three independent measurements (Supplemental Table 5).</span>



== RNA capping using Vaccinia Capping Enzyme and Cap 2´-O-Methyltransferase&nbsp; ==
<div>[https://pubmed.ncbi.nlm.nih.gov/27368341/ https://pubmed.ncbi.nlm.nih.gov/27368341/]<br/></div><div>[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5753152/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5753152/]<br/></div><div><br/></div><div>Add 7 ul 32 mM SAM to 100 ul 10X&nbsp;Capping Buffer (Final SAM is 2 mM)</div><div><br/></div><div>Combine uncapped RNA and nuclease-free water in a final volume of 14.0 μl. (Refer to step 1 in the notes on use).</div><div><br/></div><div>Heat at 65°C for 5 minutes (Refer to step 2 in the notes on use).</div><div><br/></div><div>Place tube on ice for 5 minutes.</div><div><br/></div><div>Add the following components in the order specified:</div><div>Denatured uncapped RNA (from above) 14.0 μl</div><div>10X Capping Buffer with 2 mM SAM: 2.0 μl</div><div>GTP (10 mM) 1.0 μl</div><div><s>SAM (4 mM, dilute 32 mM stock to 4 mM) 1.0 μl</s></div><div>Vaccinia Capping Enzyme (10 U/μl) 1.0 μl</div><div>mRNA Cap 2´-O-Methyltransferase (50 U/μl)&nbsp; 1.0 μl</div><div>RNase Inhibitor, Murine 0.5&nbsp;μl</div><div>Total: 20.0 μl</div><div>Note: Use of RNase Inhibitor is recommended to enhance stability of RNA in the reaction. Add 0.5 μl of RNase Inhibitor (e.g., Murine RNase Inhibitor NEB #M0314) during reaction set up. Subtract the additional volume from the amount of H2O used in the reaction.</div><div><br/></div><div>Incubate at 37°C for 60 min<br/></div><div>(For RNA less than 200 nt long increase incubation time to 2 hours).</div><div><br/></div><div>&nbsp;</div><div>RNase Inhibitor, Murine M0314S40 units/μl 3,000 units $73.00</div><div><br/></div>
IVT reactions: 1 µM primers, 40 mM Tris pH 8, 5 mM DTT, 1 mM spermidine, 0.01% Triton X-100, 4 mM each of NTP in the RNA sequence, 0.2 µM T7 RNA polymerase, 20–60 mM MgCl2.

=== RNA purification using anion-exchange chromatography ===

RNA from IVT can be purified using anion exchange chromatography on a 22 × 250 mm DNAPac PA100 column (Dionex) operated at 80°C.&nbsp;[https://www.thermofisher.com/order/catalog/product/043011#/043011 https://www.thermofisher.com/order/catalog/product/043011#/043011]

The column is run in buffers containing 20 mM Tris pH 8.0 and 5 M Urea (pH adjusted to 8.0 at room temperature). RNA of different lengths is eluted with a NaCl gradient. Salt gradients can be designed according to the respective RNA lengths to be separated. Short RNAs (2–10 nt) typically elute between 50 and 200 mM NaCl while longer constructs (60–80 nt) elute at 300–400 mM NaCl. RNA containing fractions can be analyzed using urea-PAGE, pooled and precipitated&nbsp; as below. After precipitation, RNAs can be desalted using a prepacked PD-10 Desalting column (GE Healthcare) and subsequently concentrated using a speedvac device.

=== RNA precipitation ===

RNA can be precipitated with 0.3 M NaOAc pH 5.2 and 0.7 volumes of isopropanol at −20°C for at least 2 h. The reaction is then centrifuged at 9000g and −5°C for 1 h and the pellet is washed with cold 75% ethanol and centrifuged again for 30 min. The resulting pellet is air dried and resuspended in the desired buffer for further&nbsp; applications. For short RNAs (2–10 nt), precipitation can be&nbsp;carried out with 0.2 M NaCl and 3.5 volumes of EtOH (replacing the NaOAc and isopropanol) and a precipitation time of 12 h

=== RNA capping ===

Capping reactions is performed in buffer containing 50 mM Tris, pH 8, 5 mM KCl, 1 mM MgCl2, and 1 mM DTT with 20 µM RNA. 0.5 mM GTP, 0.1 mM SAM, and capping enzyme according to the previously determined substrate to enzyme ratio is added. The reaction is incubated at 37°C and prepared for analysis by Urea-PAGE by adding an equal volume of sample buffer containing 8 M Urea, 20 mM EDTA, 2 mM Tris pH 8, and Bromophenol blue and Xylene Cyanol.

For cap-1:&nbsp; cap-1 RNA, Vaccinia MTase (NEB) is added to the capping reaction

RNA promoter sequence Promoter sequence T7 promoter Φ6.5 (for start with G) CGTAATACGACTCACTATAGG T7 promoter Φ2.5 (for start with A) CGTAATACGACTCACTATTAG

== Cas13a ==

In vitro transcription of Cas13a. Plasmids were linearized with Not-I HF (New England Biolabs) overnight at 37 °C. Linearized templates were purified by sodium acetate (Thermo Fisher Scientific) precipitation and rehydrated with nuclease-free water. In vitro transcription was performed overnight at 37 °C using the HiScribe T7 Kit (NEB) following the manufacturer’s instructions (N1-methyl-pseudouridine modified). The resulting RNA was treated with DNase I (Aldevron) for 30 min to remove the template, and it was purified using lithium chloride precipitation (Thermo Fisher Scientific). The RNA was heat denatured at 65 °C for 10 min before capping with a Cap-1 structure using guanylyl transferase and 2′-O-methyltransferase (Aldevron). mRNA was then purified by lithium chloride precipitation, treated with alkaline phosphatase (NEB) and purified again. mRNA concentration was measured using a Nanodrop. mRNA stock concentrations were 1–3 mg ml−1. Purified mRNA products were analyzed by gel electrophoresis to ensure purity. crRNA guides were purchased from Integrated DNA Technologies or GenScript [https://www.aldevron.com/hubfs/content/Documents/ald-IVT-enzyme-tech-guide-090010916.pdf https://www.aldevron.com/hubfs/content/Documents/ald-IVT-enzyme-tech-guide-090010916.pdf]

== Enzymes for mRNA research ==

[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4986899/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4986899/]

T7 RNA Polymerase 9135-130MCG 1

2598 pET29-Turbo-T7-RNAPo

E coli Inorganic Pyrophosphatase 9132-350MCG 1

2259 need to do cloning

DNase I 9136-70MCG 1&nbsp;

202 pET28-DNase1 need to subclone into pMBP vector

Ribonuclease Inhibitor 9134-180MCG 1

[http://18.206.161.119/eln/index.php/623_625_637_1494_1499_1734_1735_1740_1747_1750 pT7A30-RNaseInhibitor Sus scrofa (wild pig) RNase Inhibitor 1499]&nbsp;

Guanylyltransferase 9131-300MCG 2

2'-O-Methyltransferase 9130-1450MCG 2 An E. coli strain that carries the gene for the Vaccinia mRNA Cap 2´-O-Methyltransferase. Poly(A) Polymerase 9133-430MCG 2

Ribonuclease Inhibitor 9134-660MCG 2

Guanylyltransferase Catalyzes addition of 7-methylguanylate cap structure (Cap 0) to the 5’ end of RNA in three steps:


<div>T7 RNA Polymerase 2598 pET29-Turbo-T7-RNAPo</div><div>E coli Inorganic Pyrophosphatase&nbsp; 2259&nbsp;&nbsp;</div><div>Ribonuclease Inhibitor&nbsp; pT7A30-RNaseInhibitor Sus scrofa (wild pig)&nbsp; RNase Inhibitor 1499&nbsp;</div><div>Guanylyltransferase&nbsp; 2943</div><div>2'-O-Methyltransferase 2753</div><div>E coli&nbsp; Poly(A) Polymerase&nbsp; &nbsp; 2773</div><div>Yeast Poly(A) Polymerase&nbsp; 940 pET28b-yPolyA-Pol</div><div><br/></div><div><br/></div>
2’-O-Methyltransferase Reactions 1 & 2: Converts Cap 0 to Cap 1 by adding a methyl group to the ribose 2’-OH of the first nucleotide adjacent to the cap structure. Activity can be measured using the EPIgeneousTM Methyltransferase Assay (Cisbio).

Reaction 3: Conversion of an RNA substrate pppN(pN)n to GpppN(pN)n with increasing amounts of enzyme can be visualized by gel electrophoresis (uncapped, lower band; capped, upper band).

Methyl transfer from SAM to GpppN(pN)n producing SAH and Cap 0 mRNA can be measured using the EPIgeneousTM Methyltransferase Assay (Cisbio).

[https://www.aldevron.com/products/ivt-enzyme-kits https://www.aldevron.com/products/ivt-enzyme-kits]

IVT Buffer: [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC306405/pdf/nar00265-0210.pdf https://www.ncbi.nlm.nih.gov/pmc/articles/PMC306405/pdf/nar00265-0210.pdf]

40 mM Tris-HCl (pH 8.1 at 37°C), 1 mM spermidine, 5 mM dithiothreitol, 50 ug/ml bovine serum albumin, 0.01% (vlv) Triton X-100 and 80 mg/ml polyethylene glycol (8000 MW)

[https://www.neb.com/gmp/gmp-grade-reagents-for-rna-synthesis https://www.neb.com/gmp/gmp-grade-reagents-for-rna-synthesis]

Inorganic pyrophosphatase [https://www.neb.com/products/m0361-pyrophosphatase-inorganic-e-coli https://www.neb.com/products/m0361-pyrophosphatase-inorganic-e-coli] PPase is prepared from an E. coli strain containing a clone of the E. coli inorganic pyrophosphatase gene.

DNaseI&nbsp;: [https://www.neb.com/products/m0303-dnase-i-rnase-free https://www.neb.com/products/m0303-dnase-i-rnase-free] An E. Coli strain that carries an MBP fusion clone of Bovine Pancreatic DNase I.



== Flp mRNA ==

PCR&nbsp; using these two primers

Template; 2895 T7-VEE-NLS-Flp

T7-HBG-F<br/>TAATACGACTCACTATAAGGACATTTGCTTCTGACACAAC<br/>VEE-pA-R GAATTAATTCCCCTCGAcgcgttctag

sequence

T7 (modified)<br/>TAATACGACTCACTATAAGG&nbsp;&nbsp;<br/>ACATTTGCTTCTGACACAAC<br/>TGTGTTCACTAGCAACCTCAAACAATT<br/>Kozak<br/>GCCACC<br/>ATGGCCCCCAAGAAAAAGCGGAAAGTGATGAGCCAGTTCGACATCCTGTGCAAGACCCCCCCCAAGGTGCTCGTGCGGCAGTTCGTGGAAAGATTCGAGAGGCCCAGCGGCGAGAAGATCGCCTCTTGTGCCGCCGAGCTGACCTACCTGTGCTGGATGATCACCCACAACGGCACCGCCATCAAGCGGGCCACCTTCATGAGCTACAACACCATCATCAGCAACAGCCTGAGCTTCGACATCGTGAACAAGAGCCTGCAGTTCAAGTACAAGACCCAGAAGGCCACCATCCTGGAAGCCAGCCTGAAGAAGCTGATCCCCGCCTGGGAGTTTACCATCATCCCCTACAACGGCCAGAAGCACCAGAGCGACATCACCGATATCGTGTCCAGCCTGCAGCTGCAGTTCGAGAGCAGCGAGGAAGCCGACAAGGGCAACAGCCACAGCAAGAAGATGCTGAAGGCCCTGCTGAGCGAGGGCGAGAGCATCTGGGAGATCACAGAGAAGATCCTGAACAGCTTCGAGTACACCAGCCGGTTCACCAAGACCAAGACCCTGTACCAGTTCCTGTTCCTGGCCACCTTTATCAACTGCGGCCGGTTCTCCGACATCAAGAACGTGGACCCCAAGAGCTTCAAGCTGGTGCAGAACAAGTACCTGGGCGTGATCATTCAGTGCCTCGTGACCGAGACAAAGACCAGCGTGTCCCGGCACATCTACTTTTTCAGCGCCAGAGGCCGGATCGACCCCCTGGTGTACCTGGACGAGTTCCTGAGAAACAGCGAGCCCGTGCTGAAGAGAGTGAACCGGACCGGCAACAGCAGCTCCAACAAGCAGGAATACCAGCTGCTGAAGGACAACCTCGTGCGGTCCTACAACAAGGCCCTGAAGAAAAACGCCCCCTACCCCATCTTCGCCATTAAGAACGGCCCCAAGTCCCACATCGGCCGGCACCTGATGACCAGCTTTCTGAGCATGAAGGGCCTGACAGAGCTGACCAACGTCGTGGGCAATTGGAGCGACAAGAGGGCCTCTGCCGTGGCCAGAACCACCTACACCCACCAGATCACAGCCATCCCCGACCACTACTTCGCCCTGGTGTCTCGGTACTACGCCTACGACCCCATCAGCAAAGAGATGATCGCCCTGAAGGACGAGACAAACCCCATCGAGGAATGGCAGCACATCGAGCAGCTGAAGGGCAGCGCCGAGGGCAGCATCAGATACCCTGCCTGGAACGGCATCATCTCCCAGGAAGTGCTGGACTACCTGAGCAGCTACATCAACCGGCGGATCTAG

Not1&nbsp;<br/>GCGGCCGC<br/>AAGCTGCTTACATAGAACTCGCGGCGATTG<br/>Sph1<br/>GCATGC

CGCCTTAAAATTTTTATTTTATTTTTtCTTTTCTTTTCCGAATCGGATTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAAAACGCGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAttaatAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAttaatAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA<br/>PacI&nbsp;<br/>ttaattaa

Xba1<br/>tctaga

Mlu1<br/>acgcgT CGAGGGGAATTAATTC<br/>&nbsp;(VEE-pA-R GAATTAATTCCCCTCGAcgcgttctag )
[[Category:Pages with broken file links]]

https://www.targetmol.com/compound/mrtx-1719

== EGFP mRNA Protocol ==

to make template for Cleancap by PCR

T7AG-HBG-F GTTAATACGACTCACTATAagGTCTTCTGGTCCCCACAGAC--> this is for cleancap

VEE-pA-R GAATTAATTCCCCTCGAcgcgttctag

T7-HBA2-F is not as good as T7AG-HBG-F&nbsp;&nbsp;

Template: <span style="color:#FF0000;">'''<span style="background-color:#FFFF00;">2885&nbsp;</span>'''</span>T7-VEE-EGFP

(F primer T7AG-HBG-F GTTAATACGACTCACTATAagGTCTTCTGGTCCCCACAGAC- for cleancap)

sequence, 1113 bp

GTTAATACGACTCACTATAag TCTTCTGGTCCCCACAGACTCAGAGAGAACCC Kozak gccacc

EGFP atggtgTCTaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa Not1 GCGGCCGC AAGCTGCTTACATAGAACTCGCGGCGATTG

Sph1

GCATGC

CGCCTTAAAATTTTTATTTTATTTTTtCTTTTCTTTTCCGAATCGGATTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAAAACGCGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAttaatAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAttaatAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

PacI

ttaattaa

Xba1

tctaga

Mlu1

acgcgT CGAGGGGAATTAATTC

(R primer VEE-pA-R GAATTAATTCCCCTCGAcgcgttctag )

=== Cy5-UTP ===

[[File:Cy5-UTP.png|RTENOTITLE]]

IVT:&nbsp;Use 1 ul cy5-UTP for 80 ul reaction, 4 ul of buffer , 4 ul of each NTP, 4 ul enzyme, react 24 hours

[https://www.apexbt.com/cy5-utp.html, 25ul https://www.apexbt.com/cy5-utp.html,&nbsp;25ul] (10mM) $165.00,&nbsp;Cy5-UTP Catalog No. B8333

[https://www.perkinelmer.com/product/cyanine-5-utp-nel583001ea Cyanine https://www.perkinelmer.com/product/cyanine-5-utp-nel583001ea&nbsp;Cyanine] 5-UTP, 100 nmol,&nbsp;NEL583001EA 577.00 USD

[https://www.enzolifesciences.com/ENZ-42506/cyanine-5-utp-enhanced/ ENZ-42506 https://www.enzolifesciences.com/ENZ-42506/cyanine-5-utp-enhanced/&nbsp;&nbsp;ENZ-42506] 250 nmol 344.00 USD

[https://promocell.com/product/cy5-utp/ PK-PFCY5-UTP-10 https://promocell.com/product/cy5-utp/&nbsp;PK-PFCY5-UTP-10] 10 µl $ 299.00

[https://www.trilinkbiotech.com/cyanine-5-aminoallyluridine-5-triphosphate.html https://www.trilinkbiotech.com/cyanine-5-aminoallyluridine-5-triphosphate.html]

[https://www.cytivalifesciences.com/en/us/shop/molecular-biology/nucleic-acid-microarrays-and-accessories/amersham-cydye-fluorescent-nucleotides-p-06187#order https://www.cytivalifesciences.com/en/us/shop/molecular-biology/nucleic-acid-microarrays-and-accessories/amersham-cydye-fluorescent-nucleotides-p-06187#order]

PA55026Cy5-UTP 100 nmol 598.00 USD

[https://www.scbt.com/p/cyanine-5-utp Cyanine https://www.scbt.com/p/cyanine-5-utp&nbsp;Cyanine] 5-UTP, 250 nmolsc-499469,&nbsp; 250 nmol $316.00

[[File:2885 cy5-EGFP-mRNA.jpg|RTENOTITLE]]

[[File:2885 cy5-EGFP-mRNA 2.jpg|RTENOTITLE]]



=== NEB Protocol: ===

4 ul of each: 100 mM ATP, GTP, UTP, CTP, 3.2 ul of 100mM&nbsp;Cleancap, 4 ul of 10X Buffer, 40 ul of 2885 PCR product (2 ug total), 1 ul of 10mM Cy5-UTP (santa cruz), 5 ul of T7 RNA Polymerase Mix, Water to 80 ul, reaction for 24 hours.

Purify using RNAeazy spin column

Yield:&nbsp; 150 ul of 1.7 ug/ul, 50 ul of 816ng/ul

Total yield:&nbsp; 255 ug + 40 ug= 295 ug

=== Lucigen AmpliScribe High Yield Transcription Kits Protocol ===

x µL Nuclease-Free Water,

Sterile 1 µg linearised template DNA with T7 promoter

2 µL AmpliScribe T7 10X Reaction Buffer

1.5 µL 100 mM ATP

1.5 µL 100 mM CTP

1.5 µL 100 mM GTP

1 µL 100 mM UTP

6 µL 4 mM Cy5-UTP

2 µL 100 mM DTT

0.5 µL RiboGuard RNase Inhibitor

2 µL AmpliScribe T7 RNA Polymerase

20 µL Total reaction volume

Incubate at 37 °C for 2 hours

==  ==