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Team:Alberta/Notebook/BasePlasmids
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==Base Plasmid Version 1== | ==Base Plasmid Version 1== | ||
| + | Our original base plasmids contain a Kanamycin resistance cassette (p1003) bracketed by BsaI or BfuAI | ||
| + | or BbsI cut sites. If cut with either BsaI or BfuAI or BbsI, the Kanamycin cassette is release with sticky | ||
| + | ends characteristic of an A or a B BioByte. | ||
| + | |||
| + | ===BsaI plasmids=== | ||
| + | May 10, 2010: PCRed p1003 (Kanamycin cassette)with primers PrA_p1003+ and PrB'_p1003-</p> | ||
| + | May 10, 2010 PCRed p1003 (Kanamycin cassette)with primers PrB_p1003+ and PrA'_p1003-</p> | ||
| + | May 27, 2010 Digested pSB1C3 and our PCR products of p1003 (Kanamycin cassette)with Not1 </p> | ||
| + | May 27, 2010 Ligated PCR products of p1003 (Kanamycin cassette) and pSB1C3 </p> | ||
| + | May 27, 2010 Transformed Ligation. | ||
| + | May 28, 2010 Success! We got colonies that grew on Kan/Chlor plates | ||
| + | May 30, 2010-June 9,2010 Miniprep of colonies, Digested with EcoRI and XbaI to determine orientation | ||
| + | of Kanamycin Cassette until found plasmids in which the BioBrick prefix and suffix remained intact | ||
| + | |||
| + | ===BbsI Plasmids=== | ||
| + | June 10, 2010 PCRed p1003 (Kanamycin cassette)with primers PrA.Bbs_p1003+ and PrB'Bbs_p1003-</p> | ||
| + | June 10, 2010 PCRed p1003 (Kanamycin cassette)with primers PrB.Bbs_p1003+ and PrA'Bbs_p1003-</p> | ||
| + | June 14, 2010 Digested PCR product and pSB1C3 with NotI, ligated | ||
| + | June 16, 2010 Transformed from ligation</p> | ||
| + | June 17, 2010 Success! We did manage to get colonies that grew on Kan/Chlor plates</p> | ||
| + | June 18-21, 2010 Miniprep of colonies, Digested with EcoRI and XbaI to determine orientation of | ||
| + | Kanamycin Cassette until found plasmids in which the BioBrick prefix and suffix remained intact</p> | ||
| + | |||
| + | ===BfuAI Plasmids=== | ||
| + | June 15, 2010 PCRed p1003 (Kanamycin cassette)with primers PrA.Bfu_p1003+ and PrB'Bfu_p1003- | ||
| + | June 15, 2010 PCRed p1003 (Kanamycin cassette)with primers PrB.Bfu_p1003+ and PrA'Bfu_p1003- | ||
| + | 22/06/10 | ||
| + | Digested both kanamycin fragments and pSB1C3 with NotI. Ligated pSB1C3 to each of the kanamycin | ||
| + | fragments. Transformed the ligations into cells and plated on kanamycin and chloramphenicol plates for | ||
| + | selection. | ||
| + | 23/06/10 | ||
| + | Got colonies on plates from the transformations. Set up overnight liquid cultures of 8 white colonies | ||
| + | from each ligation. (red colonies are psb1C3) | ||
| + | 24/06/10 | ||
| + | Minipreped the liquids set up the night before. Digested each miniprep with XbaI and PstI. Also digested | ||
| + | each miniprep with just Pst. | ||
| + | 25/06/10 | ||
| + | Ran a gel of yesterday’s digests. For the plasmid to be correct it needed a 3kb for the single digest and a | ||
| + | 2kb and 1kb band for the double digests. 1 of the AB kanamycin part minipreps was correct and three of | ||
| + | BA kanamycin part minipreps. | ||
| + | 28/06/10 | ||
| + | The correct minipreps were nanodropped and the two with the highest concentrations were kept. | ||
===Base Plasmid Version 1.2 (Abandoned)=== | ===Base Plasmid Version 1.2 (Abandoned)=== | ||
===Base Plasmid Version 1.3 (Abandoned)=== | ===Base Plasmid Version 1.3 (Abandoned)=== | ||
Revision as of 06:06, 25 October 2010
Base Plasmid Version 1
Our original base plasmids contain a Kanamycin resistance cassette (p1003) bracketed by BsaI or BfuAI or BbsI cut sites. If cut with either BsaI or BfuAI or BbsI, the Kanamycin cassette is release with sticky ends characteristic of an A or a B BioByte.
BsaI plasmids
May 10, 2010: PCRed p1003 (Kanamycin cassette)with primers PrA_p1003+ and PrB'_p1003-</p> May 10, 2010 PCRed p1003 (Kanamycin cassette)with primers PrB_p1003+ and PrA'_p1003-</p> May 27, 2010 Digested pSB1C3 and our PCR products of p1003 (Kanamycin cassette)with Not1 </p> May 27, 2010 Ligated PCR products of p1003 (Kanamycin cassette) and pSB1C3 </p> May 27, 2010 Transformed Ligation. May 28, 2010 Success! We got colonies that grew on Kan/Chlor plates May 30, 2010-June 9,2010 Miniprep of colonies, Digested with EcoRI and XbaI to determine orientation of Kanamycin Cassette until found plasmids in which the BioBrick prefix and suffix remained intact
BbsI Plasmids
June 10, 2010 PCRed p1003 (Kanamycin cassette)with primers PrA.Bbs_p1003+ and PrB'Bbs_p1003-</p> June 10, 2010 PCRed p1003 (Kanamycin cassette)with primers PrB.Bbs_p1003+ and PrA'Bbs_p1003-</p> June 14, 2010 Digested PCR product and pSB1C3 with NotI, ligated June 16, 2010 Transformed from ligation</p> June 17, 2010 Success! We did manage to get colonies that grew on Kan/Chlor plates</p> June 18-21, 2010 Miniprep of colonies, Digested with EcoRI and XbaI to determine orientation of Kanamycin Cassette until found plasmids in which the BioBrick prefix and suffix remained intact</p>
BfuAI Plasmids
June 15, 2010 PCRed p1003 (Kanamycin cassette)with primers PrA.Bfu_p1003+ and PrB'Bfu_p1003- June 15, 2010 PCRed p1003 (Kanamycin cassette)with primers PrB.Bfu_p1003+ and PrA'Bfu_p1003- 22/06/10 Digested both kanamycin fragments and pSB1C3 with NotI. Ligated pSB1C3 to each of the kanamycin fragments. Transformed the ligations into cells and plated on kanamycin and chloramphenicol plates for selection. 23/06/10 Got colonies on plates from the transformations. Set up overnight liquid cultures of 8 white colonies from each ligation. (red colonies are psb1C3) 24/06/10 Minipreped the liquids set up the night before. Digested each miniprep with XbaI and PstI. Also digested each miniprep with just Pst. 25/06/10 Ran a gel of yesterday’s digests. For the plasmid to be correct it needed a 3kb for the single digest and a 2kb and 1kb band for the double digests. 1 of the AB kanamycin part minipreps was correct and three of BA kanamycin part minipreps. 28/06/10 The correct minipreps were nanodropped and the two with the highest concentrations were kept.
