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== '''Cloning of 1654 pT7A3-T3 DNA ligase''' == '''Purpose:''' Cloning of 1654 pT7A3-T3 DNA ligase '''Date: '''Jan , 2013<br> '''Notebook: ''' '''Page:''' '''Clone ID: 1654''' '''Experimenters: '''Shanyi == '''Results''' == === contamination by accident(1532-4)<br> === === Cloning === PCR<br> [[Image:1532 1538-2 1560 1561-1562 1563pcr.JPG]]<br> Nde1 + Xho1<br> === Colony screen === [[Image:1529 1532 1538digest.JPG]] === Cloning === PCR using contaminated T3 DNA ligase plasmid DNA as template and the following primers New_T3_DNA_Lig-Nde1-F A1 cacgcaCATATGATGAACATCTTTAACACC T3DNALigase-30 CTCGGAACTTCTCGAAGCTAGGGTGTCGCAGGGAGCCGTCCGGGGTCGCTTCCA<br>T3DNALigase-31 CTAGCTTCGAGAAGTTCCGAGGAACTGAGGACAACCCTCAAGAGAAAATGTAAC<br>T3DNALigase-32 TGATCGCTCGAGTTACATTTTCTCTTGAGGGTTGT<br> [[Image:1654pcr.JPG]]<br> digest with Nde1 + Xho1<br> [[Image:Digest for1285 1535 1654.JPG]] <br> === Colony screen === digest with Nde1 + Xho1<br> [[Image:1654digest.JPG]][[Image:1654-1 -2 -6DIGEST.JPG]]<br> SEND 1654-3 TO SEQUENCE == Protein Expression == 2013/3/29<br> SHANYI [[Image:1097 1138 1458 1494 1505 1513 1558 1562 1573 1593 1654NEW.jpg]]<br> {| 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> | PageRuler Plus Prestained protein ladder<br> | <br> | <br> | <br> | <br> | <br> | <br> |- | 1<br> | pT7A3-T4-G32<br> | T4-G32<br> | 1097<br> | 5 ml,<br> Nico21(DE3)plysN | 0.2 mM IPTG RT o/n<br> | Ni beads<br> | 4.82 / 33506.43 |- | 2<br> | pT7A3-MBP-(3c)-MutM<br> | MBP-(3c)-MutM<br> | 1138<br> | 5 ml,<br> Nico21(DE3)plysN<br> | 0.2 mM IPTG RT o/n <br> | Ni beads<br> | 8.68 / 30290.07+43.6 <br> |- | 3<br> | pT7A3-KOD<br> | KOD<br> | 1458<br> | 5 ml,<br> Nico21(DE3)plysN<br> | 0.2 mM IPTG RT o/n <br> | Ni beads <br> | 7.53 / 90022.42 |- | 4<br> | pT7A3-UDG<br> | UDG<br> | 1494<br> | 5 ml,<br> Nico21(DE3)plysN<br> | 0.2 mM IPTG RT o/n <br> | Ni beads<br> | 5.45 / 27174.73 |- | 5<br> | pT7A3-T7-Endo1<br> | T7-Endo1<br> | 1505<br> | 5 ml,<br> Nico21(DE3)plysN<br> | 0.2 mM IPTG RT o/n <br> | Ni beads<br> | 9.54 / 17171.84 <br> |- | 6<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> |- | 7<br> | pT7A3-T7-Exo<br> | T7-Exo<br> | 1558<br> | 5 ml, Nico21(DE3)plysN<br> | 0.2 mM IPTG RT o/n <br> | Ni beads<br> | 4.93 / 34502.20 |- | 8<br> | pT7A3-MBP-(3C)-Exo-1<br> | MBP-(3C)-Exo-1<br> | 1562<br> | 5 ml, Nico21(DE3)plysN<br> | 0.2 mM IPTG RT o/n <br> | Ni beads<br> | 8.80 / 18143.72<br> |- | 9<br> | pT7A3-MBP-(3C)-RnaseH<br> | MBP-(3C)-RnaseH<br> | 1573<br> | 5 ml, Nico21(DE3)plysN<br> | 0.2 mM IPTG RT o/n <br> | Ni beads<br> | 8.43 / <br> 17596.99 +43.6 |- | 10<br> | pT7A3-MBP-(3c)-E_coli_Endo IV<br> | MBP-(3c)-E_coli_Endo IV<br> | 1593<br> | 5 ml, Nico21(DE3)plysN<br> | 0.2mM IPTG RT o/n <br> | Ni beads<br> | 5.43 / <br> 31479.53 +43.6 |- | 11<br> | pT7A3-T3 DNA ligase<br> | T3 DNA ligase<br> | 1654<br> | 5 ml, Nico21(DE3)plysN<br> | 0.2 mM IPTG RT o/n<br> | Ni beads<br> | 5.14 / 39350.70 <br> |} <br> == '''Protein Purification''' == 1. Step gradient on 10 ml HisTrap column Buffer A: 10 mM imidazole, 20 mM Tris-HCl pH 8.0 , 500 mM NaCl, 0.05 mM EDTA, 1 mM beta-ME <br>Buffer B: 500 mM imidazole, 20 mM Tris-HCl pH 8, 300 mM NaCl, 0.05 mM EDTA, 1 mM beta-ME <br>method 64<br> SDS-PAGE: 2013/4/11 [[Image:1458 1654 1697after first purification.jpg]]<br> {| width="200" cellspacing="1" cellpadding="1" border="1" |- | lane<br> | colspan="2" | 1<br> | 2<br> | colspan="2" | 3<br> | colspan="2" | 4<br> | 5 <br> | 6 <br> | 7 <br> | 8 <br> | 10<br> | 11<br> | 12<br> |- | clone ID | colspan="5" | 1458<br> | colspan="2" | Marker<br><br> | colspan="4" | 1654<br> | colspan="4" | 1697<br> |- | name<br> | colspan="5" | pT7A3-KOD<br> | colspan="2" | <br> | colspan="4" | pT7A3-T3 DNA ligase<br> | colspan="4" | pCold-GST-(3c)-wt3C<br> |- | host<br> | colspan="5" | <br>Nico21DE3plysN<br> | colspan="2" | <br><br> | colspan="4" | Nico21DE3plysN<br> | colspan="4" | Nico21DE3plysN<br> |- | induction<br> | colspan="5" | 0.2 mM IPTG 25oC o/n<br> | colspan="2" | <br> | colspan="4" | 0.2 mM IPTG 25oC o/n <br> | colspan="4" | 0.2 mM IPTG 25oC o/n<br> |- | purification<br> | colspan="5" | 10ml HisTrap HP<br> | colspan="2" | <br> | colspan="4" | 10ml HisTrap HP<br> | colspan="4" | 10ml HisTrap HP<br> |- | MW<br> | colspan="5" | 7.53 / 90022.42 <br> | colspan="2" | <br> | colspan="4" | 5.14 / 39350.70<br> | colspan="4" | 21KD/8.47<br> |} Combine fraction 31-68, concentrate to 20ml<br> 2. Gel filtration on TSK 250 gel filtration. Buffer: 50 mM Na-Phosphate, pH 7.4, 200 mM NaCl<br> SDS-PAGE: 2013-4-17 [[Image:1458after2ndNi 1654aftergel filtration 1711 1712small.jpg]]<br> {| width="685" cellspacing="1" cellpadding="1" border="1" |- | lane<br> | colspan="2" | 1<br> | 2<br> | 3<br> | 4<br> | 5<br> | colspan="2" | 6<br> | 7 <br> | 8 <br> | 9 <br> | 10 <br> | colspan="2" | 11 <br> | 12<br> |- | clone ID | colspan="6" | 1458<br> | colspan="2" | Marker<br><br> | colspan="4" | 1654 <br> | colspan="2" | 1711 <br> | colspan="2" | 1712 <br> |- | name<br> | colspan="6" | pT7A3-KOD<br> | colspan="2" | <br> | colspan="4" | pT7A3-T3 DNA ligase<br> | colspan="2" | pET28b-hINFA2 h<br> | colspan="2" | pLambda-hINFA2<br> |- | host<br> | colspan="6" | Nico21DE3plysN<br> | colspan="2" | <br><br> | colspan="4" | Nico21DE3plysN<br> | colspan="2" | <br> | colspan="2" | <br> |- | induction<br> | colspan="6" | 0.2 mM IPTG 25oC o/n<br> | colspan="2" | <br> | colspan="4" | 0.2 mM IPTG 25oC o/n<br> | colspan="2" | <br> | colspan="2" | <br> |- | purification<br> | colspan="6" | 10ml HisTrap HP(2nd time)<br> | colspan="2" | <br> | colspan="4" | Bio-Sil TSK 250<br> | colspan="2" | <br> | colspan="2" | <br> |- | MW<br> | colspan="6" | 7.53 / 90022.42 <br> | colspan="2" | <br> | colspan="4" | 5.14 / 39350.70<br> | colspan="2" | <br> | colspan="2" | <br> |} concentrate fraction 28 ~31using spin concentator 3. Dialyze twice overnight.<br> Dialysis Buffer:20 mM Tris-HCl pH7.5, 50 mM NaCl, 50% Glycerol , 0.1 mM EDTA<br> 4. Store in a buffer (20 mM Tris-HCl pH7.5, 50 mM NaCl, 50% Glycerol, 0.1 mM EDTA, 1mM DTT) at -80oC (T7ligase box). <br> Concentration : 2 mg/ml<br> [[Category:Cloning]]
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