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Cleancap mRNA Invitro Transcription
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== <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 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 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 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 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 μL</span><br/> | <br/> | <br/> |- | <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">CleanCap AG, 100 mM </span><br/> | <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">7.5 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 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. 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 </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 '''<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;"> (for pCDNA5 plasmid) </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''' (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 </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;">with Flag Tag and polyA: use '''<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;"> 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;"> 2711-IRES-F</span> <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">ACGATGATAATATGGCCAGCTAGCCACCATGTTCGTCTTC </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;">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''' (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 </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 ''' '''T7-mRNA-F3'''<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;"> </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:''' 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 <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 <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>
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