Editing Cleancap mRNA Invitro Transcription (section)

== 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