Team:Caltech/Week 3
From 2011.igem.org
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==June 30== | ==June 30== | ||
- | <p>Miniprep 5 K145001 cultures and send them off for sequencing | + | <p>Miniprep 5 K145001 cultures and send them off for sequencing<br/> |
Make glycerol stocks of mCherry ([http://partsregistry.org/Part:BBa_J06702 J06702]), Lac Promoter ([http://partsregistry.org/Part:BBa_R0010 R0010]), double terminator ([http://partsregistry.org/Part:BBa_B0014 B0014] and [http://partsregistry.org/Part:BBa_B0015 B0015]) and Tet Promoter ([http://partsregistry.org/Part:BBa_R0040 R0040])<br/> | Make glycerol stocks of mCherry ([http://partsregistry.org/Part:BBa_J06702 J06702]), Lac Promoter ([http://partsregistry.org/Part:BBa_R0010 R0010]), double terminator ([http://partsregistry.org/Part:BBa_B0014 B0014] and [http://partsregistry.org/Part:BBa_B0015 B0015]) and Tet Promoter ([http://partsregistry.org/Part:BBa_R0040 R0040])<br/> | ||
- | Repeat PCR of B0014 and perform PCR to linearize backbone vector <br/> | + | Repeat PCR of B0014 and perform PCR to linearize backbone vector (pSB4A5) <br/> |
- | Digest PCR products with DpnI and purify< | + | Digest PCR products with DpnI and purify</p> |
- | + | ===Results=== | |
- | + | [[File: 630pcrgel.png|thumb|PCR of parts for pNT001 and pNT002: 1 & 2 [http://partsregistry.org/Part:BBa_B0014 B0014]; 3 & 4 [http://partsregistry.org/Part:pSB4A5 pSB4A5]; 5 blank; 6 ladder]] | |
+ | B0014 appeared in the gel. But [http://partsregistry.org/Part:pSB4A5 pSB4A5] did not. We are redoing PCR of [http://partsregistry.org/Part:pSB4A5 pSB4A5] with primers to add the prefix and suffix with longer annealing and elongating times. | ||
+ | <div><div> | ||
+ | ==July 1== | ||
+ | <p>Dpn1 digest and purification of [http://partsregistry.org/Part:pSB4A5 pSB4A5] PCR<br/> | ||
+ | Run a gel of [http://partsregistry.org/Part:pSB4A5 pSB4A5] PCR<br/> | ||
+ | Gibson assemble pNT001 and pNT002 and negative controls following Joe's protocol.<br/> | ||
+ | Transform pNT001 and pNT002 into competent cells<br/> | ||
+ | Check sequencing of K145001<br/> | ||
+ | Check if enrichment cultures are ready for transfer</p> | ||
+ | ==July 2== | ||
+ | <p>Check plates from Gibson transformations. Do colony PCR if necessary<br/> | ||
+ | Check if enrichment cultures are ready for transfer</p> | ||
+ | }} |
Revision as of 05:47, 1 July 2011
Project |
June 26Start overnight cultures of T7 polymerase ([http://partsregistry.org/Part:BBa_K145001 K145001]), mCherry ([http://partsregistry.org/Part:BBa_J06702 J06702]), Lac Promoter ([http://partsregistry.org/Part:BBa_R0010 R0010]), double terminator ([http://partsregistry.org/Part:BBa_B0014 B0014] and [http://partsregistry.org/Part:BBa_B0015 B0015]) and Tet Promoter ([http://partsregistry.org/Part:BBa_R0040 R0040]) June 27Miniprep of T7 polymerase ([http://partsregistry.org/Part:BBa_K145001 K145001]), mCherry ([http://partsregistry.org/Part:BBa_J06702 J06702]), Lac Promoter ([http://partsregistry.org/Part:BBa_R0010 R0010]), double terminator ([http://partsregistry.org/Part:BBa_B0014 B0014] and [http://partsregistry.org/Part:BBa_B0015 B0015]) and Tet Promoter ([http://partsregistry.org/Part:BBa_R0040 R0040]) and submit for sequencing ResultsMiniprep
We found growth in one of our BPA enrichment cultures, and slight growth in our ethinyl estradiol cultures. We transferred these vials today. June 28Send off continued forward sequencing for HER ResultsSequencing: All biobricks showed correct sequence except T7 Polymerase ![]() PCR of parts for pNT001 and pNT002(BioBricks + primers for Gibson): From left to right: 1 ladder; 2 blank; 3 [http://partsregistry.org/Part:BBa_R0010 R0010]; 4 [http://partsregistry.org/Part:BBa_K123000 K123000]; 5 [http://partsregistry.org/Part:BBa_B0014 B0014]; 6 [http://partsregistry.org/Part:BBa_R0040 R0040]; 7 [http://partsregistry.org/Part:BBa_K123001 K123001]; 8 [http://partsregistry.org/Part:BBa_B0015 B0015] Ran a gel of the PCR products. Strangely, lanes 3 and 4 show no DNA. We will repeat the PCR of lanes 3-5 and Gibson assemble pNT001 ([http://partsregistry.org/Part:BBa_R0010 R0010], [http://partsregistry.org/Part:BBa_K123000 K123000], [http://partsregistry.org/Part:BBa_B0014 B0014]) later in the week.
June 29
Redo PCR of [http://partsregistry.org/Part:BBa_R0010 R0010] and [http://partsregistry.org/Part:BBa_K123000 K123000] ResultsEstrogen receptor ([http://partsregistry.org/Part:BBa_K123003 K123003]) sequencing is back. Again, the translation is right, but the bases don't match. The stop codon is not in this read. We will keep sequencing.
June 30Miniprep 5 K145001 cultures and send them off for sequencing ResultsB0014 appeared in the gel. But [http://partsregistry.org/Part:pSB4A5 pSB4A5] did not. We are redoing PCR of [http://partsregistry.org/Part:pSB4A5 pSB4A5] with primers to add the prefix and suffix with longer annealing and elongating times. July 1Dpn1 digest and purification of [http://partsregistry.org/Part:pSB4A5 pSB4A5] PCR July 2Check plates from Gibson transformations. Do colony PCR if necessary
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