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== Cloning == <div>=======</div><div>3135 pCMV5-His-VH-IgG2b, His heavy</div><div>subclone from: 3135-3 pMiniT2</div><div>Vector Name: 3129 pCMV5-HA-VH-mIgG2b</div><div>Restriction Enzymes: BsrG1-Kpn1</div><div>Insert Length: 539</div><div>===</div><div>His Tag light</div><div>3001 pCMV5-His-VL-kappa</div><div><span style="color:#FF0000;">gene synthesis (not completed yet)</span><br/></div><div>==========</div><div>3151 pCMV5-Myc-H-IgG2b, <span style="color:#FF0000;">will not work</span></div><div>will not translate, need to fix sequence from BamH1-->EcoR1</div><div>subclone from 3003-H-1-PJET-1</div><div>Restriction Enzymes: BamH1-Kpn1</div><div>Vector Name: 2492 pCMV5-mIgG2b-HC</div><div>====</div><div>3075 pCMV5-Myc-L-kappa, completed</div><div>subclone from: 3003 pJet1.2-Myc 3003-L-5 light chain</div><div>Vector Name: 2493 pCMV5-mIgG-kLC</div><div>Restriction Enzymes: BamH1-Kpn1</div><div>Antibiotics: amp</div><div>====</div><div>for Myc tag Heavy, need to do 3076</div><div>3076 pCMV5-Myc-H-IgG2b (replaced by 3151)</div><div>PCR cloning from </div><div>Vector Name: 3129 pCMV5-HA-VH-mIgG2b</div><div>PCR from 3003 pJet1.2-Myc heavy</div><div>3076-myc-BsrG1-F</div><div>atcatcTGTaCActccGAGGTGGACCTGGTTGAGTC</div><div><br/></div><div>3076-myc-Nhe1-Kpn1-R</div><div>(CCACAGTGACCGTTTCTTCTGCTAGC GGTACC gatgat)</div><div>atcatcGGTACCGCTAGCAGAAGAAACGGTCACTGTGG</div><div>Restriction Enzymes: BsrG1-Kpn1: </div><div>Insert Length: 403</div><div>=====</div><div><br/></div><div>3009 pCMV5-Flag-H-IgG2, Flag heavy</div><div>subclone from: 3009-4-pMiniT2</div><div>Vector Name: 2492 pCMV5-mIgG2b-HC</div><div>Restriction Enzymes: BamH1-Kpn1</div><div>Insert Length: 539</div><div>Antibiotics: amp</div><div><br/></div><div>====</div><div>3010 pCMV5-Flag-kappa, Flag light</div><div>need to fix from 3135-4-pMiniT2 (this 3135-4 clone is actually 3010) as-F_E12.ab1 template</div><div>it is actually 3010: missing </div><div>5' end ATCATCGGATCCGCC</div><div>3' end: GGAGGAACAAAGCTCGAGATCAAGCGGGCTAGCGGTACCATCATC</div><div>using these oligos</div><div> 3010 use 3135-4 as template<br/>Positive control<br/>3010-Flag-VL-3<br/>3010-Flag-VL-10 Full fragment:<br/>-20 oC : 100 uM<br/>5--> +45 ul H2O==> 10 uM<br/>1 ul of 10 uM dilute to 20 ul H2O 3010-Flag-VL-1 F1 1 ul of 10 uM<br/>3010-Flag-VL-2 R2 1 ul of 0.5 uM <br/>3010-Flag-VL-3 F3 1 ul of 0.5 uM<br/>3010-Flag-VL-10 1 ul of 0.5 uM<br/>3010-Flag-VL-11 1 ul of 0.5 uM<br/>3010-Flag-VL-12 1 ul of 10 uM </div><div><br/></div><div>Vector Name: 2493 pCMV5-mIgG-kLC</div><div>Restriction Enzymes: BamH1-Kpn1</div><div><br/></div><div>=======</div><div>3129 pCMV5-HA-VH-mIgG2b, heavy</div><div>Vector Name: 2492 pCMV5-mIgG2b-HC</div><div><br/></div><div>finished:</div><div>PCR from 3011 pJet1.2-HA Heavy chain</div><div><br/></div><div><br/></div><div>3129-BamH1-F</div><div>atcatcggatccAAGATCTAGAGCCGCCACCATG</div><div><br/></div><div>3129-Kpn1-F</div><div>atcatcGTACCagacacggtaaccagtgttc</div><div>(gaacactggttaccgtgtctGGTACgatgat)</div><div>Clone into pMiniT2</div><div><br/></div><div>Then subclone into 2492 pCMV5-mIgG2b-HC</div><div>Restriction Enzymes: BamH1 - Kpn1</div><div>==========</div><div>3130 pCMV5-HA-VL-mIgKappa HA light</div><div>Vector Name: 2493 pCMV5-mIgG-kLC</div><div>PCR from 3011 pJet1.2-HA light chain</div><div>clone: 3011-L-1 </div><div><br/></div><div>3130-BamH1-R</div><div>atcatcggatccGCCGCCACCATGGACATGAGGGTC</div><div><br/></div><div><br/></div><div>3130-Kpn1-R</div><div>atcatcGGTACCccgcttcagttccagcttggttccgccgccaaatg</div><div>(catttggcggcggaaccaagctggaactgaagcggGGTACC gatgat)</div><div><br/></div><div>Restriction Enzymes: BamH1-Kpn1</div><div>-------</div><div<br/></div><div>'''3127 pET28b-qBeta-qBeta-6XHis'''<br/></div><div>Primers</div><div>3127-Nde1-F<br/>gatgat CATATGg GCCAAACTGGAAACCGTTAC<br/>3127-HindIII-R<br/>(gatcagctgaatccggcgtat AAGCTT atcatc)<br/>gatgatAAGCTTatacgccggattcagctgatc<br/></div><div>Insert size: 402</div><div>Vector: 3097 pET28b-qBeta<br/>PCR using these primers and 3097 pET28b-qBeta as template,<br/></div><div>digest with Nde1-HindIII, clone into Nde1-HindII digested 3097 pET28b-qBeta<br/></div><div><br/></div><div><br/></div><div></div><div><br/></div> {| border="1" cellspacing="1" cellpadding="1" style="width:500px;" |- | <s>ID</s> | <s>Name</s> | <s>DNA Size</s> | <s>Vector</s> | <s>Method</s> | <s>Status</s><br/> | <s>Date</s> | <s>Enzyme</s><br/> | <s>Expression</s> | <s>Note</s> |- | <s>940</s><br/> | <s>pET28b-yPolyA-Pol</s><br/> | <s>1713</s> | <s>pET28b</s> | <s> subclone from 2015 pEC2 using Nde1 -Xho1</s> | <s>completed</s> | <br/> | <br/> | <br/> | <br/> |- | <s>3105</s><br/> | <s> pET28b-AaLS</s><br/> | <s>699</s> | <br/> | | <s>finished</s> | 10/18/17 | <span style="font-family: Arial, Verdana, sans-serif; font-size: 12px; background-color: rgb(251, 251, 239);">Nco1+ Xho1</span> | has expression | <br/> |- | 3027<br/> | pCEH0-mIgG2b-HC<br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> |- | <s>3097</s><br/> | <s> pET28b-Qbeta</s><br/> | <s>720</s> | <br/> | <br/> | <s>3097-2 sequence OK, subcloned into Nco1 + Sal1 into pET28b (Nco1+ Xho1)</s><br/> | <br/> | pJet1.2: Nco1+ Xho1 Vector: Nco1+Sal1 | no his tag | <br/> |- | 3127<br/> | pET28b-qBeta-qBeta-6XHis<br/> | <br/> | <br/> | <br/> | PCR qbeta and clone back into 3097 | <br/> | Nde1-HindIII | C-His tag | <br/> |- | <s>3098</s><br/> | <s> pET28b-AP205</s><br/> | <s>592</s> | <br/> | <br/> | <s>good, subcloned into pET28b, no his tag, digestion with BamH1 to get c_his tag</s> | <s>transform Nico to test expression</s> | <s>Nco1+ </s>Xho1<br/> | <br/> | <br/> |- | 2490<br/> | pMiniT-mIgG2a-HC<br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> |- | 2491<br/> | pCMV5-mIgG-kappa<br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> |- | <s>3003</s><br/> | <s> pJet1.2-Myc</s><br/> | <br/> | <br/> | <br/> | <s>submitted sequencing, light chain in pJet1.2 is good,</s> <s>finished</s> <s>need to do subcloning</s> | 10/18/17 | <br/> | <br/> | <br/> |- | 812<br/> | pET30b-phoA<br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> |- | <s>3082</s><br/> | <s> pET28b-Cel1</s> <s>947 pT7A30-6His-pMBP</s> | <s>837</s> | <br/> | <br/> | <s>pJet1.2, one clone sequencing verified,</s> <s>subcloned into pET28b failed, toxic gene</s> | <s>10/18/17</s> | <s>Nde1 + X</s>ho1 | toxic in pET28b | <br/> |- | <span style="font-family: Arial, Verdana, sans-serif; font-size: 12px; white-space: nowrap;">3074</span><br/> | <span style="font-family: Arial, Verdana, sans-serif; font-size: 12px; white-space: nowrap;">pET30b-HA-avitag-BirA</span><br/> | <br/> | <br/> | <br/> | in pJet1.2 is good, subclone into pET28b, no his tag, need to subclone into pT7T30-Avi-BirA (still no his tag) | <br/> | <br/> | <br/> | <br/> |- | <s>3011</s> | <s>pJet1.2-HA</s> | <br/> | <br/> | <br/> | <s>H & L in pJet1.2 done, need to sublcone</s> | 10/10/2017 | <br/> | <br/> | <br/> |- | 3010<br/> | pJet1.2-Flag<br/> | <br/> | <br/> | <br/> | not working yet, need to do gene synthesis | <br/> | <br/> | <br/> | <br/> |- | <s>3003</s> | <s>pCMV5-Myc-HC</s><br/> | <br/> | <br/> | <br/> | <s>pJet1.2 H & L sequenced</s><br/> | <br/> | <br/> | <br/> | <span style="color:#FF0000;">mAb 9E10 c-terminal c-myc tag</span><br/> |- | <span style="color:#FF0000;">3001</span> | <span style="color:#FF0000;">pCMV5-His-VL-kappa</span> | <br/> | <br/> | <br/> | <span style="color:#FF0000;">problem, can not get a good band from gene synthesis, should send it to IDT</span> | <br/> | <br/> | <br/> | <br/> |- | 3135 <br/> | pCMV5-His-VH-IgG2b<br/> | <br/> | <br/> | <br/> | pMiniT2 completed | <br/> | <br/> | <br/> | <br/> |- | 938 | PNGase F, | 1145 | pET30b<br/> | Nde1-Xho1 | pJet1.2, sequencing is good, need to subclone into pET30b | <br/> | <br/> | <br/> | C-His<br/> |- | 939 | Tte-Exo III | 780 | pET30b<br/> | Nde1-Xho1<br/> | pJet1.2 sequencing is good, need to subclone into pET30b | <br/> | <br/> | <br/> | C-His |- | 942 | VVN endonuclease | 716 | pET28b-pMBP<br/> | Nco1-Xho1 | pJet1.2 bad quality, need to restransform and make plasmid for sequencing | <br/> | <br/> | <br/> | C-His<br/> |- | 3122 | Crm197, secreted | 1743 | pET30b<br/> | Nde1-Xho1<br/> | pJet1.2<br/> | <br/> | <br/> | <br/> | C-His |- | 931 | PAK16D ligase | 2267 | pET28b | Nde1-HindIII-Xho1<br/> | pJet1.2 fragment 2 is good fragment 1 is also good | <br/> | <br/> | <br/> | N-His |- | 945<br/> | pT7A30-pMBP<br/> | <br/> | pT7A3 | Xba1-Nco1<br/> | PCR from 1281 pMAL-p5E or 1282 pMAL-p5E-AvPAL | <br/> | <br/> | <br/> | <br/> |- | 947<br/> | pT7A30-6His-pMBP<br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> |- | <s>948</s><br/> | <s>pJet1.2-ccdb</s><br/> | <br/> | <br/> | <br/> | <s>sequenced, completed</s> | <br/> | <br/> | <br/> | <br/> |- | 944 | pET30b-pfu-exo-<br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> |- | 949<br/> | pET28b-GST-sfGFP-Myc-HA-Flag-V5<br/> | <br/> | 433 546<br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> |- | 3075<br/> | pCMV5-Myc-L-kappa<br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> |- | 3076 | pCMV5-Myc-H-IgG2b<br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> |- | 3010<br/> | pCMV5-Flag-kappa<br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> |- | 3009 | pCMV5-Flag-H-IgG2b<br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> |- | 3129<br/> | pCMV5-HA-VH-mIgG2b<br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> |- | 3130 <br/> | 2493 pCMV5-HA-VL-mIgKappa<br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> |- | 3131 <br/> | pCEL0-HA-VL-mIgKappa<br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> |- | 3132 <br/> | pCEH0-HA-VH-mIgG2b<br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> |- | <br/> | pCMV-PD1-AviTag-c6His<br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> |- | <s>940</s> | <s>940 pET28b-yPolyA-Pol</s><br/> | <br/> | <br/> | <br/> | <s>sequenced, completed</s> | <br/> | <br/> | <br/> | <br/> |- | 824<br/> | pET28b-VVTopo<br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> | <br/> |- | <br/> | <span style="color:#FF0000;">3107 pFastbac-Cel1</span><br/> | <br/> | <br/> | <br/> | GGATCC<br/>ACCATGAAGTTCTTGGTGAACGTGGCGCTGGTGTTCATGGTCGTGTACATCTCCTACATTT ACGCGTGGGGCAAAGAAGGCCACTACGCCGTGTGTAAGATTGCGGAGGGCTTTTTGTCGGA AGACGCATTGGGAGCGGTCAAAGCCTTGTTGCCGGACTACGCGGAAGGCGACTTGGCAGCC GTATGTAGCTGGGCCGACGAGATCAGACACAACTTTCACTGGAGATGGTCGGGCCCACTGC ATTACGTCGACACGCCGGATTACAGATGCAACTACGAGTACTGCCGCGACTGTCACGACTT CAGAGGCCACAAAGACATTTGCGTCACGGGCGCGATATACAACTACACGAAACAATTGACG TCGGGCTACCACAACAGTGGCTCCGAGATTCGATACAACCTCACGGAGGCCTTGATGTTCC TCTCGCATTTCATTGGCGACGTGCACCAACCGCTGCATGTGGGCTTTACGGGCGATGAAGG CGGAAATACGATCATTGTCCGTTGGTACCGCAGAAAGACCAACCTCCACCACATATGGGAC AACATGATCATCGACTCGGCGTTGAAGACCTACTACAACAGCGACCTGGCCATAATGATCC AGGCGATTCAAAGAAACATCACCGGCGATTGGTCCTTTGACATCAGCAGCTGGAAGAACTG TGCCAGTGACGACACTGCTTGTCCGAACCTATACGCGTCGGAGAGCATCTCGTTGGCCTGT AAATTTGCCTACAGAAATGCCACCCCCGGTTCGACGCTGGGCGACGACTACTTCTTGTCGC GATTGCCGATTGTTGAAAAACGCCTCGCCCAAGGCGGTATTAGATTGGCCGCCACCTTGAA CCGTATTTTTGCCTCGCAACCGAAAATCTCGC GAAACACGAAGACAAGAGAG CGAGAAG ACGACGCCGGTAGACTACATCGAGTGGTCGCCAT TGCAACAGT TCAGCGGAAGCCACCACC ATCACCACCATCATCACGGC<br/>ctcgag CAT CAT CAT CAT CAT CAC<br/>TGATAA AAGCTT | <br/> | <br/> | <br/> | <br/> |}
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