In vitro transcription using PCR product

PCR product containing T7-IRES and can be cloned into pCDNA5-FRT

1. PCR T7-IRES from  956 pT7CFE1-CHis  

Primers:

IVT-T7-F AACGACGGCCAGTGAATTGTAATAC

IVT-IRES-R

 CATGGTGGTGGCCATATTATC 
(GTACCACCACCGGTATAATAG)

PCR T7-IRES from  956 pT7CFE1-CHis  

PCR GOI 

GOI-F (with Nhe1 site )

CACGATGATAATATGGCCAGCTAGC caccatgAGAACTCAATTA

GOI-R (with Kpn1 and polyA)

(TTCTTCAGGTTGGACAGCTTAAgcttaaaaaaaaaaaaaa)

ttttttttttttttaagcTTAAGCTGTCCAACCTGAAGAA

Then combined two PCR products, PCR again

IVT-T7-F + GOI-R.

Use this PCR product for making mRNA: does not need to use capped analog

1. PCR T7-IRES from  956 pT7CFE1-CHis  

Primers:

IVT-T7-F AACGACGGCCAGTGAATTGTAATAC

IVT-IRES-R

 CATGGTGGTGGCCATATTATC 
(GTACCACCACCGGTATAATAG)

IVT-Flpo-F GATAATATGGCCACCACCATGGCTCCCAAGAAGAAGAGGAAGGTGATG

IVT-Flpo-R

 TTTTTTTTTTTTTTTTTTTTTCTAAATCCGCCTGTTGATGTAGC
(AAAAAAAAAAAAAAAAAAAAAGATTTAGGCGGACAACTACATCG)

PCR product is 566 bp T7-IRES, T7 Promoter (TAATACGACTCACTATAGGG

AACGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAATTAATTCCGGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCACCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCACCACCATG

template: 956 pT7CFE1-CHis

2. PCR target gene (GOI) such as Flpo

IVT-Flpo-F: overlap sequence          Flpo 

GATAATATGGCCACCACCATG  

IVT-Flpo-R:

TTT TTT TTT TTT TTT TTT TTT CTA AATCCGCCTGTTGATGTAGC
(GCTACATCAACAGGCGGATT TAG AAA AAA AAA AAA AAA AAA AAA)

Template: 3277 pMiniT-Flpo, 

3. PCR again using purified T7-IRES + GOI

IVT-T7-F + IVT-Flpo-R.

PCR Product Sequence

AACGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAATTAATTCCGGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCACCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAA CACGATGATAATATGGCCACCACC

ATGAGCCAGTTCGACATCCTGTGCAAGACCCCCCCCAAGGTGCTGGTGCGGCAGTTCGTGGAGAGATTCGAGAGGCCCAGCGGCGAGAAGATCGCCAGCTGTGCCGCCGAGCTGACCTACCTGTGCTGGATGATCACCCACAACGGCACCGCCATCAAGAGGGCCACCTTCATGAGCTACAACACCATCATCAGCAACAGCCTGAGCTTCGACATCGTGAACAAGAGCCTGCAGTTCAAGTACAAGACCCAGAAGGCCACCATCCTGGAGGCCAGCCTGAAGAAGCTGATCCCCGCCTGGGAGTTCACCATCATCCCTTACAACGGCCAGAAGCACCAGAGCGACATCACCGACATCGTGTCCAGCCTGCAGCTGCAGTTCGAGAGCAGCGAGGAGGCCGACAAGGGCAACAGCCACAGCAAGAAGATGCTGAAGGCCCTGCTGTCCGAGGGCGAGAGCATCTGGGAGATCACCGAGAAGATCCTGAACAGCTTCGAGTACACCAGCAGGTTCACCAAGACCAAGACCCTGTACCAGTTCCTGTTCCTGGCCACATTCATCAACTGCGGCAGGTTCAGCGACATCAAGAACGTGGACCCCAAGAGCTTCAAGCTGGTGCAGAACAAGTACCTGGGCGTGATCATTCAGTGCCTGGTGACCGAGACCAAGACAAGCGTGTCCAGGCACATCTACTTTTTCAGCGCCAGAGGCAGGATCGACCCCCTGGTGTACCTGGACGAGTTCCTGAGGAACAGCGAGCCCGTGCTGAAGAGAGTGAACAGGACCGGCAACAGCAGCAGCAACAAGCAGGAGTACCAGCTGCTGAAGGACAACCTGGTGCGCAGCTACAACAAGGCCCTGAAGAAGAACGCCCCCTACCCCATCTTCGCTATCAAGAACGGCCCTAAGAGCCACATCGGCAGGCACCTGATGACCAGCTTTCTGAGCATGAAGGGCCTGACCGAGCTGACAAACGTGGTGGGCAACTGGAGCGACAAGAGGGCCTCCGCCGTGGCCAGGACCACCTACACCCACCAGATCACCGCCATCCCCGACCACTACTTCGCCCTGGTGTCCAGGTACTACGCCTACGACCCCATCAGCAAGGAGATGATCGCCCTGAAGGACGAGACCAACCCCATCGAGGAGTGGCAGCACATCGAGCAGCTGAAGGGCAGCGCCGAGGGCAGCATCAGATACCCCGCCTGGAACGGCATCATCAGCCAGGAGGTGCTGGACTACCTGAGCA

GCTACATCAACAGGCGGATTTAGAAA AAA AAA AAA AAA AAA AAA

MSQFDILCKTPPKVLVRQFVERFERPSGEKIASCAAELTYLCWMITHNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKKLIPAWEFTIIPYNGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESIWEITEKILNSFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGVIIQCLVTETKTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEYQLLKDNLVRSYNKALKKNAPYPIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWSDKRASAVARTTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQLKGSAEGSIRYPAWNGIISQEVLDYLSSYINRRI

4. IVT

Other methods

mRNAs were transcribed to contain 110 nt (pLuc19) or 101 nt (pTEV-VRC01-LC-A101 and pTEV-VRC01-HC-A101) poly(A) tails. m1Ψ-5′-triphosphate (TriLink) instead of UTP was used to generate modified nucleoside-containing mRNA. RNAs were capped using the m7G capping kit with 2′-O-methyltransferase (ScriptCap, CellScript) to obtain cap1