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== '''Cloning of 1123 pT7A3-MBP-(3C)-T4-RNA-Lig2''' == '''Purpose:''' Cloning of 1123 pT7A3-MBP-(3C)-T4-RNA-Lig2<br> '''Date: '''Jan 4, 2013<br> '''Notebook: ''' '''Page:''' '''Clone ID: '''1123 '''Experimenters: '''Jennifer Truong == '''Results''' == === Cloning === Date: Jan 4, 2013<br> PCR using 1uL of 1:10 dilution of T4 genomic DNA and the following primers<br> T4RNA-lig-Nde1-F<br>atcgac CATatg tttaaaaagtatagcagtc T4RNA-lig-Xho1-R<br>(cttggattgagttggtaagttaaCTCGAG atcgaa)<br>ttcgatCTCGAGttaacttaccaactcaatccaag<br> See lane 1 [[Image:1123 1045 Amp.JPG]] Cut out band, and purify Digest insert (lane ) and vector (1514 pT7A3-MBP-(3C) lane ) with RE Nde1 and Xho1 Cut out bands, ligate together and transform <br> === Screening === == '''Result intepretation/Summary'''<br> == == '''Conclusion''' == <br> <br> == '''Cloning of 1123 pT7A3-MBP-(3C)-T4-RNA-Lig2''' == '''Purpose:''' Cloning of 1123 pT7A3-MBP-(3C)-T4-RNA-Lig2<br> '''Date: '''Jan 4, 2013<br> '''Notebook: ''' '''Page:''' '''Clone ID: '''1123 '''Experimenters: '''Shanyi == '''Results''' == === Cloning === PCR [[Image:1123 1045pcr.JPG]] <br> Cut out band, and purify Digest insert (lane ) and vector (1514 pT7A3-MBP-(3C) lane ) with RE Nde1 and Xho1 [[Image:1049 1097 1123DIGEST-right.JPG]]4.92 / 24309.87 Cut out bands, ligate together and transform PCR<br> [[Image:1045 1049 1122 1123pcr.JPG]]<br> [[Image:Digest for 1044 1049 1122 1123 1 16 2013.JPG]]<br> <br> === Screening === [[Image:1123 1558digest 1 11 2013.JPG]]<br> send 1123-2 to sequeng, it is 1026. send 1123-4 to sequence again.<br> [[Image:1122 1123DIGEST.JPG]] == <br> == == '''Result intepretation/Summary'''<br> == == '''Conclusion''' == <br> == Protein Expression == 2013/3/11<br> [[Image:1049 1123 1501 1503 1508 1512 1513 1537 1557 1572.jpg]] {| width="832" cellspacing="1" cellpadding="1" border="1" |- | Lane<br> | Name<br> | Protein Name<br> | Clone ID<br> | Host<br> | Induction<br> | Purification<br> | pI/Mw<br> |- | M<br> | Invitrogen SeeBlue plus2<br> | <br> | <br> | <br> | <br> | <br> | <br> |- | 1<br> | pT7A3-T4_RNA_Ligase_1<br> | T4_RNA_Ligase_1<br> | 1049<br> | 5 ml,<br> Nico21(DE3)plysN | 0.2 mM IPTG RT o/n<br> | Ni beads<br> | 4.63 / 47485.21 <br> |- | 2<br> | pT7A3-MBP-(3C)-T4-RNA-Lig2<br> | T4-RNA-Lig2<br> | 1123<br> | 5 ml,<br> Nico21(DE3)plysN<br> | 0.2 mM IPTG RT o/n <br> | Ni beads<br> | 5.11 / 39375.95 +43.6 <br> |- | 3<br> | pT7A3-Klenow<br> | Klenow<br> | 1501<br> | 5 ml,<br> Nico21(DE3)plysN<br> | 0.2 mM IPTG RT o/n <br> | Ni beads <br> | 5.11 / 71294.91 <br> |- | 4<br> | pT7A3-T4_RNA_Ligase_ 2_Truncated<br> | T4_RNA_Ligase_ 2_Truncated<br> | 1503<br> | 5 ml,<br> Nico21(DE3)plysN<br> | 0.2 mM IPTG RT o/n <br> | Ni beads<br> | 5.14 / 29824.85 <br> |- | 5<br> | pT7A3-DNA-Pol-1<br> | DNA-Pol-1<br> | 1508<br> | 5 ml,<br> Nico21(DE3)plysN<br> | 0.2 mM IPTG RT o/n <br> | Ni beads<br> | 5.40 / 103118.12<br> |- | 6<br> | pT7A3-Phi29DNAPol<br> | Phi29DNAPol<br> | 1512<br> | 5 ml, Nico21(DE3)plysN<br> | 0.2 mM IPTG RT o/n <br> | Ni beads<br> | 8.61 / 66714.05 <br> |- | 7<br> | pT7A3-polyAPol<br> | polyAPol<br> | 1513<br> | 5 ml, Nico21(DE3)plysN<br> | 0.2 mM IPTG RT o/n <br> | Ni beads<br> | 9.67 / 53870.91 <br> |- | 8<br> | pT7A3-Klenow(3'->5'_exo–)<br> | Klenow(3'->5'_exo–)<br> | 1537<br> | 5 ml, Nico21(DE3)plysN<br> | 0.2 mM IPTG RT o/n <br> | Ni beads<br> | 5.77 / 67969.77 <br> |- | 9<br> | pT7A3-MBP-(3C)-Lambda-Exo<br> | Lambda-Exo<br> | 1557<br> | 5 ml, Nico21(DE3)plysN<br> | 0.2 mM IPTG RT o/n <br> | Ni beads<br> | 5.36 / 25925.62 +43.6<br> |- | 10<br> | pT7A3-MBP-(3c)-Endonuclease VIII<br> | Endonuclease VIII<br> | 1572<br> | 5 ml, Nico21(DE3)plysN<br> | 0.2 mM IPTG RT o/n <br> | Ni beads<br> | 7.77 / 29845.13+43.6 <br> |} <br> <br> [[Category:Cloning]]
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