MRNA Products
| Product Name | Plasmid Name | Cat # | Clone # | Plasmid E coli stock | mRNA Length | Digestion or PCR | Note |
| Cre | T7-VEE-T7AG_Cre |
2893 |
35/F4 |
1610 bp |
T7 AG TAATACGACTCACTATAAGG VEE-pA-R GAATTAATTCCCCTCGAcgcgttctag 1610 bp |
||
| Cre | pT7AG-Cre |
2942 |
? |
T7AG TAGTAATACGACTCACTATAAGG VEE-pA-R GAATTAATTCCCCTCGAcgcgttctag 1613 bp |
|||
| Fluc | T7-VEE-FLuc |
2889 | 35/F1 |
||||
| Fluc | pT7AG-Fluc-pA |
2952 |
35/F1 |
T7AG-HBG-F GTTAATACGACTCACTATAagGTCTTCTGGTCCCCACAGAC or T7AG TAATACGACTCACTATAAGG + VEE-pA-R GAATTAATTCCCCTCGAcgcgttctag |
|||
| RFP | T7-VEE-TagRFP |
2904 |
35/F5 |
T7AG-HBG-F GTTAATACGACTCACTATAagGTCTTCTGGTCCCCACAGAC--> this is for cleancap VEE-pA-R GAATTAATTCCCCTCGAcgcgttctag, |
|||
| 8000nt mRNA, self amplify TagGFP2 mRNA | T7-VEE-TagGFP2-E3L-8000 |
2951 |
33/I1 |
8770 | Xba1, Mlu1, PacI | for thermo project | |
| EGFP mRNA | T7-VEE-EGFP |
EG-307, EG-320 |
2885 |
35/F7 |
1093, 8642 |
PCR using T7AG-HBG-F GTTAATACGACTCACTATAagGTCTTCTGGTCCCCACAGAC--> this is for cleancap product with pA and T7 promoter
|
not SAM, if by PCR ==[edit]Digestion with Xba1, Mlu1, PacI: make SAM EGFP mRNA |
| EGFP mRNA ARCA |
T7AG-HBG-F TAATACGACTCACTATAAGGtcttctggtccccacagac (not the best primer) VEE-pA-R GAATTAATTCCCCTCGAcgcgttctag 1149 bp |
||||||
| T7AG-EGFP |
2953 |
? |
T7AG-HBG-F TAATACGACTCACTATAAGGtcttctggtccccacagac VEE-pA-R GAATTAATTCCCCTCGAcgcgttctag 1149bp |
||||
| T7-VEE-TagGFP2 |
2926 | ? | |||||
| FLP self amplifying mRNA | T7-VEE-NLS-Flp |
EG-1044 EG-1045 |
2895 |
33/I3 |
1724, 9283 |
to make template by PCR for EG-1044
T7-NLS-FLPe TAATACGACTCACTATAGGGATGGCCCCCAAGAAAAAGCGG VEE-pA-R GAATTAATTCCCCTCGAcgcgttctag 1724 bp --- for EG-1045, digest plasmid with Xba1, Mlu1, PacI
|
not SAM, if by PCR
Digestion with Xba1, Mlu1, PacI: make SAM EGFP mRNA |
| FLuc mRNA | T7-VEE-FLuc |
EG-316 |
2889 2952 |
35/F1 |
2023 |
to make template for Cleancap by PCR, superHiFi 2889-PCR-F: + 2023 bp 2nd round, Taq T7AG-HBG-F GTTAATACGACTCACTATAagGTCTTCTGGTCCCCACAGAC + digestion: digest 2953 with Mlu1, purify DNA with glycogen + propanol == digest 2889 with Mlu1 for self amplify mRNA |
|
| NanoFluc | pRham-T7ag-NLuc-2A-EGFP |
2935 |
35/H3 |
||||
| Rluc | pT7ag-Rluc |
2934 |
35/H1 |
||||
| Rluc | T7ag-VEE-RLuc |
2946 |
35/H4 |
||||
| eSpCas9-T2A-EGFP |
2931 | can not use for mRNA production | |||||
| Cas9-EGFP, self amplify mRNA | T7-VEE-eSPCas9-EGFP |
EG-317, EG-314 |
2938 |
35/G9 |
for Cas9-EGFP SAM: digest 2938 with Mlu1 For Cas9-EGFP, PCR using T7AG-HBG-F GTTAATACGACTCACTATAagGTCTTCTGGTCCCCACAGAC + VEE-pA-R GAATTAATTCCCCTCGACGCGTTCTAG
|
EG-317: NLS-eCas9-T2A-EGFP Self-Amplifying mRNA, GSK or331295-1 EG-314: NLS-eCas9-T2A-EGFP mRNA (CleanCap), or331295-2 | |
| eCas9 self amplify mRNA | T7ag-VEE-eCas9 |
EG-322 |
2947 |
35/H5 |
digest 2947 with Mlu1, very low mRNA yield | GSK or331295-4 | |
| eCas9 cleancap |
T7ag-pA-eCas9 |
EG-321 |
2939 |
35/H2 |
digest 2939 with Mlu1 | GSK r331295-3 | |
RNA Synthesis with Modified Nucleotides (E2050)
[edit]Protocols.io also provides an https://www.protocols.io/view/RNA-Synthesis-with-Modified-Nucleotides-E2050-cv7w9m interactive version of this protocol</a> where you can discover and share optimizations with the research community.
We strongly recommend wearing gloves and using nuclease-free tubes and reagents to avoid RNase contamination. Reactions are typically 20 μl but can be scaled up as needed. Reactions should be assembled in nuclease-free microfuge tubes or PCR strip tubes.
The kit is capable of synthesizing biotin- or dye-modified RNA with the following protocol. The recommended molar ratio of modified NTP (Biotin-, Fluorescein-, Digoxigenin-, or Aminoallyl-NTP) to standard NTP is 1:2. The following reaction set-up assumes modified UTP is used. Please note that Dye- or Biotin-NTPs are not supplied with the kit.
- Thaw the necessary kit components, mix and pulse-spin in microfuge to collect solutions to the bottoms of tubes.
- Assemble the reaction at room temperature in the following order:
| Nuclease-free water | X µl |
| NTP Buffer Mix | 5 µl (5 mM each NTP final) |
| Modified UTP (10 mM) | 5 µl (2.5 mM final) |
| Template DNA | X µl (1 µg) |
| T7 RNA Polymerase Mix | 2 µl |
| Total reaction volume | 20 µl |
- Mix thoroughly, pulse-spin and incubate at 37°C for 2 hours. For short (< 300 nt) transcripts incubate at 37°C for 4–16 hours. Note that the ratio of modified nucleotide to standard nucleotide can be adjusted by varying the amount of the NTP Buffer Mix and modified nucleotide. For complete modified nucleotide substitution we recommended using the T7 High Yield RNA Synthesis Kit https://www.neb.com/products/e2040-hiscribe-t7-high-yield-rna-synthesis-kit NEB E2040, in which the four nucleotides are supplied separately.
Figure 1 shows the time course of labeled RNA synthesis using 1 µg control template with Biotin-16-UTP and Fluorescein-12-UTP following the above reaction setup.
Modified ribonucleotides reduce transcription efficiency; therefore, lower transcription yields should be expected as compared to transcription using unmodified NTPs. In general, Biotin-NTP and Aminoallyl-NTP have an insignificant effect on yields, while lower yields can be expected for transcription reactions containing Fluorescein-NTP or Cy-NTP. In addition, transcripts containing modified ribonucleotides have reduced electrophoretic mobility due to higher molecular weight.
https://www.neb.com/%7E/media/Catalog/All-Protocols/494442EBCCE947D08CDFDFBEA28E8416/Content/E2050c.jpg - Figure 1. RNA synthesis with modified nucleotides
Reactions were incubated at 37°C in a thermocycler. Transcripts were purified by spin columns and quantified on a NanoDrop Spectrophotometer. - Optional: To remove template DNA, add 30 μl nuclease-free water and 2 μl of DNase I (RNase-free), mix and incubate at 37°C for 15 minutes.
- Proceed with purification synthesized RNA (we recommend the Monarch RNA Cleanup Kits, https://www.neb.com/products/t2040-monarch-rna-cleanup-kit-50-ug NEB T2040 or https://www.neb.com/products/t2050-monarch-rna-cleanup-kit-500-ug T2050 or analysis of transcription products by gel electrophoresis.
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
Template: 2885 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 )
T7-EGFP-F: TAATACGACTCACTATAAGGAGACCATGGTGAGCAAGGGC
Cleancap, mw 1145.60 g
1145.6 g in 1 L: 1M
1145.6 mg in 1 ml: 1M
11.45 mg in 1 ml: 10 uM
11.45 mg in 100 ul: 100uM
1XIVT reaction buffer:
50 mM Tris-HCl, pH 7.5 15 mM MgCl2 5 mM dithiothreitol (DTT) 2 mM spermidine Make 10X stock and store at -20°C.
10X NTPs 25 mM each of ATP, CTP, GTP, and UTP Store at -20°C.
Inorganic pyrophosphatase
Inorganic pyrophosphatase (PPase) catalyzes the hydrolysis of inorganic pyrophosphate to form orthophosphate: P2O7-4 + H2O -> 2HPO4-2. Inorganic pyrophosphate is released when a nucleoside triphosphate is incorporated/polymerized into the growing chain. This helps to prevent against any inhibitory effect of having pyrophosphate around (i.e. prevents the "reverse" reaction.) This is an optional component of the transcription reaction. If you leave it out, often you will see something precipitate (white) in your transcription reaction. This is the pyrophosphate.
Make a 0.1 U/μL stock solution in H2O and store at -20°C.
Transcription reaction For a 100 μL reaction ("preparative scale"): 10 μL 10X transcription buffer 10 μL 10X NTPs xx μL DNA template (5–10 pmol) 5 μL inorganic pyrophosphatase (0.1 U/μL): 0.005 U/μL final concentration xx μL T7 RNAP (25 U/μL final concentration) 1 μl RNase inhibitor (Murine RNase Inhibitor, NEB, 40000 U/ml)
====
[edit]Add the desired protospacer sequence to the T7FwdVar oligo and order the oligo from your favorite oligonucleotide supplier. There are many programs available for protospacer design that attempt to optimize on- and/or off-target activity. Which program is most useful depends upon many factors including type of editing, organism being edited, etc. Choice of protospacer design program is beyond the scope of this protocol.
The transcript will start with the bolded G just 5’ of the dashes in the T7FwdVar oligo. T7 RNA polymerase requires a 5’ G for proper transcript initiation. If your protospacer has a G at the 5’ end, you can omit it from the T7FwdVar design to avoid duplication of the G. If your protospacer has a C, T, or A at the 5’ end, add the whole protospacer sequence to T7FwdVar. In this case, there will be an extra G added to the 5’ end of the protospacer, but literature indicates this will have minimal effect unless your guide is very short.
Primers: T7FwdVar oligo (5’-TAATACGACTCACTATAG--protospacer sequence—GTTTCAGAGCTATGCTGGAAAC-3’ ) T7RevLong oligo (5’-AAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTCAACTTGCTATGCTGTTTCCAGCATAGCTCTGA-3’ ) T7FwdAmp primer (5’-TAATACGACTCACTATAG-3’) T7RevAmp primer (5’-AAAAAAAGCACCGACTCGGTGC-3’ )
For eachT7FwdVar oligo you designed, set up the following PCR (total volume should be 20.0 µL). Make sure everything is RNase free and filter tips are used. Furthermore, wipe down everything (in every step of protocol) with RNase Away to ensure no contamination with RNAse.
10.6 µl DEPC-treated H2O
4.0 uL 5x Phusion HF Buffer 0.4 µl 10 mM dNTPs 0.4 µl T7FwdVar (1 µM) 0.4 µl T7RevLong (1 µM) 2 µl T7FwdAmp (10 µM) 2 µl T7RevAmp (10 µM) 0.2 µl Phusion HF DNA polymerase (2u/µl)
If making multiple sgRNA templates, prepare a master mix with all components except T7FwdVar. Include a no template control (omit T7FwdVar).
Primers: T7FwdVar oligo (5’-TAATACGACTCACTATAG--protospacer sequence—GTTTCAGAGCTATGCTGGAAAC-3’ ) T7RevLong oligo (5’-AAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTCAACTTGCTATGCTGTTTCCAGCATAGCTCTGA-3’ ) T7FwdAmp primer (5’-TAATACGACTCACTATAG-3’) T7RevAmp primer (5’-AAAAAAAGCACCGACTCGGTGC-3’ )
Run PCR:
98˚ 30 sec
98˚ 10 sec
51˚ 10 sec
72˚ 10 sec
30x steps 2-4
72˚ 2 min 4˚ hold
No PCR cleanup necessary at this point
We like to use HiScribe T7 High Yield RNA Synthesis Kit but any T7 RNA synthesis kit should be fine.
Mix the following to make 20 µl totalT7 transcriptionmix volume reagent 2 µl 10x Buffer 1x 2 µl "ATP (100 mM), 10 mM" 2 µl "GTP (100 mM), 10 mM" 2 µl "CTP (100 mM), 10 mM" 2 µl "UTP (100 mM), 10 mM" 8 µl "DNA template from Step 3, (usually ~4µg, 50 pmol)" 2 µl T7 RNA polymerase mix
Incubate transcription mix for ~18 hours (over night) at 37˚ in a thermocycler 18:00:00 START 37 °C
Remove DNA template by adding 1 µl of RNase-free DNase; incubate 15 min at 37C in thermocycler 00:15:00 37C incubationSTART DNase I (RNase-free) - 1,000 unitsNew England BiolabsCatalog #M0303S 37 °C
T7 in vitro transcription results in RNA carrying a 5'-triphosphate group. This triggers a RIG-I-mediated innate immnue response in mammalian cells and can cause cell death, particularly in primary cells. We highly recommend treating your IVT sgRNA with recombinant Shrimp Alkaline Phosphatase (rSAP) before proceeding to the purification step. We found that rSAP treatment must be rigorous to completely remove all 5-PPP groups from your RNA. However, rSAP binds tightly to RNA and NEB recommends to only use the minimal amount needed.
Add 3ul of NEB CutSmart Buffer (comes with the rSAP enzyme) Add 4µl of H2O Add 2ul (2 units) of rSAP
Shrimp Alkaline Phosphatase (rSAP) - 500 unitsNew England BiolabsCatalog #M0371S
Mix well and incubate at 37C for 3h 03:00:00 START 37 °C
sgRNAs need to be purfied before transfection. There are different methods one could purify their sgRNAs. We found that while SPRI bead clean-up of RNAs is quick and gives reliable yields (see older versions of protocol), SPRI bead purified sgRNAs still cause an elevated immune response even after Phosphatase treatment. We therefore tested different column purification kits and found that column purification of sgRNAs completely eliminates the immune response after Phosphatase treatment.
We use the Qiagen RNeasy Mini Prep Kit and follow the manufacturer's instructions with the following modifications: Yields of from column purification can be low as the sgRNAs are small and most kits are designed to bind longer RNAs. We found the Qiagen RNeasy kit works ok, but others recommend the miRNeasy kit from Qiagen. It might be worth trying a few different kits to optimize your yield.
Adjust sgRNA sample to a volume of 100 µl with RNase-free water. Add 350 µl RLT Buffer to the sample
Add 250 µl 100% ethanol and mix well by pipetting.
Tranfer the sample (~700 µl) to an RNeasy mini spin column; spin for 15 sec at >8000 g
Then transferr the remainder onto the same column; spin for 15 sec
Add 500 µl RPE Buffer; spin 15 sec
Repeated this wash step Moved spin column to a new collection tube and spin for 1 min to dry the membrane completely
Move spin column to an RNAse-free 1.5 ml microfuge tube Add 33 µl DEPC-treated H2O; spin 1 min Optional: Repeat the elution to collect any remaining RNA
Measure your RNA concentration by Nanodrop or Qubit. You can also check integrity/corrrect size of the sgRNA(s) on the RNA tapestation. Store sgRNAs at -80C
https://www.protocols.io/run/in-vitro-transcription-of-guide-rnas-and-5-triphos-bqjbmuin