Editing Capped mRNA (section)

== NEB T7 mRNA Protocol ==

Kit: T7 High Yield RNA Synthesis Kit, Cat #: E2040S



'''Plasmid Templates'''

*Digest plasmid with an enzyme to generate blunt ends (EcoRV) or 5´-overhangs (e.g., BamH1). Select site should be at the downstream of the insert to be transcribed.
*After linearization, use spin column to purify DNA. Elute with water.



'''PCR product:'''

&nbsp;100–500 ng of PCR fragments can be used in a 20 μl in vitro transcription reaction.

PCR primer design:

F: TAA TAC GAC TCA CTA TAG GG + 21 bp of the 5' end of GOI.

R: Gene specific primer.

<br/>Thaw the necessary kit components, mix and pulse-spin in a microfuge to collect solutions to bottom of tubes. Keep on ice.

Make 20 mM GTP solution by combining 2 μl of 20 mM GTP and 8 μl of nuclease-free water. Extra 20 mM GTP can be stored at -20°C for future use.<br/>Prepare cap analog at the concentration of 40 mM.<br/><br/>Assemble the reaction at room temperature in the following order:

{| cellspacing="0" cellpadding="1"
|-
| Nuclease-free water
| X μl
| &nbsp;
|-
| 10X Reaction Buffer
| 2 μl
| &nbsp;
|-
| ATP (100 mM)
| 2 μl
| 10 mM final
|-
| UTP (100 mM)
| 2 μl
| 10 mM final
|-
| CTP (100 mM)
| 2 μl
| 10&nbsp;mM final
|-
| GTP (20 mM)
| 2 μl
| 2&nbsp;mM final
|-
| Cap Analog (40 mM)
| 4 μl
| 8 mM final
|-
| Template DNA
| X μl
| 1 μg
|-
| T7 RNA Polymerase Mix
| 2 μl
| &nbsp;
|-
| '''Total reaction volume'''
| '''20 μl'''
| &nbsp;
|}

<br/>Mix thoroughly, pulse-spin and incubate at 37°C for 2 hours.<br/><br/>The yield per reaction is 40–50 μg RNA with approximately 80% capped RNA transcripts. Table 1 shows the effect of varying the ratio of cap analog to GTP on the yield of RNA. Increasing the ratio of cap analog to GTP will increase the proportion of capped RNA transcripts, however it also significantly decreases the yield of the transcription reaction. A ratio of cap analog to GTP of 4:1 is preferably used.<br/><br/>Table 1. Effect of cap analog:GTP ratios on RNA yield

{| cellspacing="0" cellpadding="1"
|-
| width="127" | '''Cap Analog: GTP Ratio'''
| width="138" | '''Concentration of Cap'''<br/>
'''Analog: GTP (mM)'''

| width="113" | '''RNA Yield (μg) in'''<br/>
'''2 hours'''

| width="97" | '''% Capped RNA'''
|-
| 0:1
| 0:10
| 180
| 0
|-
| 1:1
| 5:5
| 90–120
| 50
|-
| 2:1
| 6.7:3.3
| 60–90
| 67
|-
| 4:1
| 8:2
| 40–50
| 80
|-
| 8:1
| 8.9:1.1
| 20–25
| 89
|}

<br/>To remove template DNA, add 2 μl of DNase I (RNase-free), mix and incubate at 37°C for 15 minutes.<br/><br/>Proceed with purification of synthesized RNA using a spin column

PCR Template Preparation<br/>1. Cloning of IDT gene fragment into pMiniT2 or pJet1.2<br/>2683 (DS1)<br/>TAATACGACTCACTATAGGGAGgccAccATGAGAACTCAATTACCCCCTGCATACACTAATTCTTTCACACGTGGTGTTTATTACCCTGACAAAGTTTTCAGATCCTCAGTTTTACATTCAACTCAGGACTTGTTCTTACCTTTCTTTTCCAATGTTACTTGGTTCCATGCTATACATGTCTCTGGGACCAATGGTACTAAGAGGTTTGATAACCCTGTCCTACCATTTAATGATGGTGTTTATTTTGCTTCCACTGAGAAGTCTAACATAATAAGAGGCTGGATTTTTGGTACTACTTTAGATTCGAAGACCCAGTCCCTACTTATTGTTAATAACGCTACTAATGTTGTTATTAAAGTCTGTGAATTTCAATTTTGTAATGATCCATTTTTGGGTGTTTATTACCACAAAAACAACAAAAGTTGGATGGAAAGTGAGTTCAGAGTTTATTCTAGTGCGAATAATTGCACTTTTGAATATGTCTCTCAGCCTTTTCTTATGGACCTTGAAGGAAAACAGGGTAATTTCAAAAATCTTAGGGAATTTGTGTTTAAGAATATTGATGGTTATTTTAAAATATATTCTAAGCACACGCCTATTAATTTAGTGCGTGATCTCCCTCAGGGTTTTTCGGCTTTAGAACCATTGGTAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACTTTACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTTCTTCAGGTTGGACAGCTGGTTAGTAACCGCTGAGCAATAACTAGCAAAAAAAAAA

<br/>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 (CAAAGACGATGACGACAAGTAACTCGAGAAAAAAAAAAAAAAA)<br/>21 ul H2O<br/>+ 25 ul 2 X Phusion PCR mix (NEB)

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<br/>MRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGDYKDDDDK

purify PCR fragment using Qiagen gel purification kit

Use 1 ug for IVT