https://eln.excellgen.com/index.php?action=history&feed=atom&title=In_vitro_transcription_using_PCR_product In vitro transcription using PCR product - Revision history 2026-10-18T04:05:13Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=In_vitro_transcription_using_PCR_product&diff=20537&oldid=prev Paul.xu at 23:41, 22 April 2020 2020-04-22T23:41:06Z <p></p> <p><b>New page</b></p><div>== PCR product containing T7-IRES and can be cloned into pCDNA5-FRT ==<br /> <br /> 1. PCR T7-IRES from&amp;nbsp;&amp;nbsp;956 pT7CFE1-CHis&amp;nbsp;&amp;nbsp;<br /> <br /> Primers:<br /> <br /> IVT-T7-F&amp;nbsp;&#039;&#039;&#039;AACGACGGCCAGTGAATTGTAATAC&#039;&#039;&#039;<br /> <br /> IVT-IRES-R<br /> <br /> CATGGTGGTGGCCATATTATC <br /> (GTACCACCACCGGTATAATAG)<br /> <br /> PCR&amp;nbsp;T7-IRES from&amp;nbsp;&amp;nbsp;956 pT7CFE1-CHis&amp;nbsp;&amp;nbsp;<br /> <br /> PCR GOI&amp;nbsp;<br /> <br /> GOI-F (with Nhe1&amp;nbsp;site )<br /> <br /> &#039;&#039;&#039;CACGATGATAATATGGCCA&#039;&#039;&#039;&lt;span style=&quot;color:#008000;&quot;&gt;GCTAGC&#039;&#039;&#039;&amp;nbsp;&#039;&#039;&#039;&lt;/span&gt;caccatgAGAACTCAATTA<br /> <br /> GOI-R (with Kpn1 and polyA)<br /> <br /> (TTCTTCAGGTTGGACAGCTT&#039;&#039;&#039;AAgctt&#039;&#039;&#039;aaaaaaaaaaaaaa)<br /> <br /> ttttttttttttttaagcTTAAGCTGTCCAACCTGAAGAA<br /> <br /> Then combined two PCR products, PCR again<br /> <br /> IVT-T7-F +&amp;nbsp;GOI-R.<br /> <br /> <br /> <br /> == &#039;&#039;&#039;&lt;span style=&quot;color:#FF0000;&quot;&gt;Use this PCR product for making mRNA: does not need to use capped analog&lt;/span&gt;&#039;&#039;&#039; ==<br /> <br /> 1. PCR T7-IRES from&amp;nbsp;&amp;nbsp;956 pT7CFE1-CHis&amp;nbsp;&amp;nbsp;<br /> <br /> Primers:<br /> <br /> IVT-T7-F &#039;&#039;&#039;AACGACGGCCAGTGAATTGTAATAC&#039;&#039;&#039;<br /> <br /> IVT-IRES-R<br /> <br /> CATGGTGGTGGCCATATTATC <br /> (GTACCACCACCGGTATAATAG)<br /> <br /> IVT-Flpo-F &#039;&#039;&#039;GATAATATGGCCACCACCATG&#039;&#039;&#039;GCTCCCAAGAAGAAGAGGAAGGTGATG<br /> <br /> IVT-Flpo-R<br /> <br /> TTTTTTTTTTTTTTTTTTTTTCTAAATCCGCCTGTTGATGTAGC<br /> (AAAAAAAAAAAAAAAAAAAAAGATTTAGGCGGACAACTACATCG)<br /> <br /> <br /> <br /> PCR product is 566 bp T7-IRES, &#039;&#039;T7 Promoter (&lt;span style=&quot;color:#FF0000;&quot;&gt;TAATACGACTCACTATAGGG&lt;/span&gt;&#039;&#039;<br /> <br /> &#039;&#039;&#039;AACGACGGCCAGTGAATTG&#039;&#039;&#039;&lt;span style=&quot;color:#FF0000;&quot;&gt;&#039;&#039;&#039;&#039;&#039;TAATAC&#039;&#039;&#039;GACTCACTATAGG&#039;&#039;G&lt;/span&gt;CGAATTAATTCCGGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCACCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGAT&#039;&#039;&#039;GATAATATGGCCACCACCATG&#039;&#039;&#039;<br /> <br /> template:&amp;nbsp;956 pT7CFE1-CHis<br /> <br /> == 2. PCR target gene (GOI)&amp;nbsp;such as Flpo ==<br /> <br /> IVT-Flpo-F: overlap sequence&amp;nbsp; &amp;nbsp; &amp;nbsp; &amp;nbsp; &amp;nbsp; Flpo&amp;nbsp;<br /> <br /> &#039;&#039;&#039;GATAATATGGCCACCACCATG&amp;nbsp;&amp;nbsp;&#039;&#039;&#039;<br /> <br /> IVT-Flpo-R:<br /> <br /> TTT TTT TTT TTT TTT TTT TTT CTA AATCCGCCTGTTGATGTAGC&lt;br/&gt;(GCTACATCAACAGGCGGATT TAG AAA AAA AAA AAA AAA AAA AAA)<br /> <br /> Template:&amp;nbsp;&lt;span style=&quot;color: rgb(0, 0, 0); font-family: Arial, Verdana, sans-serif; font-size: 12px;&quot;&gt;3277&amp;nbsp;&lt;/span&gt;pMiniT-Flpo,&amp;nbsp;<br /> <br /> == 3. PCR again using purified&amp;nbsp;T7-IRES + GOI ==<br /> <br /> IVT-T7-F + IVT-Flpo-R.<br /> <br /> PCR Product Sequence<br /> <br /> &#039;&#039;&#039;AACGACGGCCAGTGAATTG&lt;u&gt;&#039;&#039;&lt;span style=&quot;color:#FF0000;&quot;&gt;TAATAC&lt;/span&gt;&#039;&#039;&lt;/u&gt;&#039;&#039;&#039;&lt;u&gt;&#039;&#039;&lt;span style=&quot;color:#FF0000;&quot;&gt;GACTCACTATAGGG&lt;/span&gt;&#039;&#039;&lt;/u&gt;CGAATTAATTCCGGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCACCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAA&amp;nbsp;&#039;&#039;&#039;CACGATGATAATATGGCCACCACC&#039;&#039;&#039;<br /> <br /> ATGAGCCAGTTCGACATCCTGTGCAAGACCCCCCCCAAGGTGCTGGTGCGGCAGTTCGTGGAGAGATTCGAGAGGCCCAGCGGCGAGAAGATCGCCAGCTGTGCCGCCGAGCTGACCTACCTGTGCTGGATGATCACCCACAACGGCACCGCCATCAAGAGGGCCACCTTCATGAGCTACAACACCATCATCAGCAACAGCCTGAGCTTCGACATCGTGAACAAGAGCCTGCAGTTCAAGTACAAGACCCAGAAGGCCACCATCCTGGAGGCCAGCCTGAAGAAGCTGATCCCCGCCTGGGAGTTCACCATCATCCCTTACAACGGCCAGAAGCACCAGAGCGACATCACCGACATCGTGTCCAGCCTGCAGCTGCAGTTCGAGAGCAGCGAGGAGGCCGACAAGGGCAACAGCCACAGCAAGAAGATGCTGAAGGCCCTGCTGTCCGAGGGCGAGAGCATCTGGGAGATCACCGAGAAGATCCTGAACAGCTTCGAGTACACCAGCAGGTTCACCAAGACCAAGACCCTGTACCAGTTCCTGTTCCTGGCCACATTCATCAACTGCGGCAGGTTCAGCGACATCAAGAACGTGGACCCCAAGAGCTTCAAGCTGGTGCAGAACAAGTACCTGGGCGTGATCATTCAGTGCCTGGTGACCGAGACCAAGACAAGCGTGTCCAGGCACATCTACTTTTTCAGCGCCAGAGGCAGGATCGACCCCCTGGTGTACCTGGACGAGTTCCTGAGGAACAGCGAGCCCGTGCTGAAGAGAGTGAACAGGACCGGCAACAGCAGCAGCAACAAGCAGGAGTACCAGCTGCTGAAGGACAACCTGGTGCGCAGCTACAACAAGGCCCTGAAGAAGAACGCCCCCTACCCCATCTTCGCTATCAAGAACGGCCCTAAGAGCCACATCGGCAGGCACCTGATGACCAGCTTTCTGAGCATGAAGGGCCTGACCGAGCTGACAAACGTGGTGGGCAACTGGAGCGACAAGAGGGCCTCCGCCGTGGCCAGGACCACCTACACCCACCAGATCACCGCCATCCCCGACCACTACTTCGCCCTGGTGTCCAGGTACTACGCCTACGACCCCATCAGCAAGGAGATGATCGCCCTGAAGGACGAGACCAACCCCATCGAGGAGTGGCAGCACATCGAGCAGCTGAAGGGCAGCGCCGAGGGCAGCATCAGATACCCCGCCTGGAACGGCATCATCAGCCAGGAGGTGCTGGACTACCTGAGCA<br /> <br /> &#039;&#039;&#039;GCTACATCAACAGGCGGATTTAGAAA AAA AAA AAA AAA AAA AAA&#039;&#039;&#039;<br /> <br /> MSQFDILCKTPPKVLVRQFVERFERPSGEKIASCAAELTYLCWMITHNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKKLIPAWEFTIIPYNGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESIWEITEKILNSFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGVIIQCLVTETKTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEYQLLKDNLVRSYNKALKKNAPYPIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWSDKRASAVARTTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQLKGSAEGSIRYPAWNGIISQEVLDYLSSYINRRI<br /> <br /> <br /> <br /> <br /> <br /> 4.&amp;nbsp;[[FLP_Recombinase_mRNA#RNA_Synthesis_Protocols|IVT]]<br /> <br /> == Other methods ==<br /> <br /> 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<br /> [[Category:Products]] [[Category:Protocols]]</div> Paul.xu