Team:Alberta/Notebook/BasePlasmids

From 2010.igem.org

(Difference between revisions)
(BsaI plasmids)
(BsaI plasmids)
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* May 27, 2010 Transformed Ligation.
* May 27, 2010 Transformed Ligation.
* May 28, 2010 Success! We got colonies that grew on Kan/Chlor plates
* 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
+
* 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
-
of Kanamycin Cassette until found plasmids in which the BioBrick prefix and suffix remained intact
+
===BbsI Plasmids===
===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 PrA.Bbs_p1003+ and PrB'Bbs_p1003
-
* June 10, 2010 PCRed p1003 (Kanamycin cassette)with primers PrB.Bbs_p1003+ and PrA'Bbs_p1003-</p>
+
* June 10, 2010 PCRed p1003 (Kanamycin cassette)with primers PrB.Bbs_p1003+ and PrA'Bbs_p1003
* June 14, 2010 Digested PCR product and pSB1C3 with NotI, ligated
* June 14, 2010 Digested PCR product and pSB1C3 with NotI, ligated
-
* June 16, 2010 Transformed from ligation</p>
+
* June 16, 2010 Transformed from ligation
-
* June 17, 2010 Success! We did manage to get colonies that grew on Kan/Chlor plates</p>
+
* June 17, 2010 Success! We did manage to get colonies that grew on Kan/Chlor plates
-
* June 18-21, 2010 Miniprep of colonies, Digested with EcoRI and XbaI to determine orientation of
+
* 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
-
Kanamycin Cassette until found plasmids in which the BioBrick prefix and suffix remained intact</p>
+
===BfuAI Plasmids===
===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 PrA.Bfu_p1003+ and PrB'Bfu_p1003
-
* June 15, 2010 PCRed p1003 (Kanamycin cassette)with primers PrB.Bfu_p1003+ and PrA'Bfu_p1003-
+
* June 15, 2010 PCRed p1003 (Kanamycin cassette)with primers PrB.Bfu_p1003+ and PrA'Bfu_p1003
-
* June 22,2010 Digested both kanamycin fragments and pSB1C3 with NotI. Ligated pSB1C3 to each of the kanamycin
+
* June 22,2010 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.
-
fragments. Transformed the ligations into cells and plated on kanamycin and chloramphenicol plates for
+
* June 23,2010 Got colonies on plates from the transformations. Set up overnight liquid cultures of 8 white colonies from each ligation. (red colonies are psb1C3)
-
selection.
+
* June 24,2010 Minipreped the liquids set up the night before. Digested each miniprep with XbaI and PstI. Also digested each miniprep with just Pst.
-
* June 23,2010 Got colonies on plates from the transformations. Set up overnight liquid cultures of 8 white colonies
+
* June 25, 2010 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.
-
from each ligation. (red colonies are psb1C3)
+
-
* June 24,2010 Minipreped the liquids set up the night before. Digested each miniprep with XbaI and PstI. Also digested
+
-
each miniprep with just Pst.
+
-
* June 25, 2010 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.
+
* June 28 2010 The correct minipreps were nanodropped and the two with the highest concentrations were kept.
* June 28 2010 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)===

Revision as of 06:13, 25 October 2010

TEAM ALBERTA

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
  • May 10, 2010 PCRed p1003 (Kanamycin cassette)with primers PrB_p1003+ and PrA'_p1003
  • May 27, 2010 Digested pSB1C3 and our PCR products of p1003 (Kanamycin cassette)with Not1
  • May 27, 2010 Ligated PCR products of p1003 (Kanamycin cassette) and pSB1C3
  • 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
  • June 10, 2010 PCRed p1003 (Kanamycin cassette)with primers PrB.Bbs_p1003+ and PrA'Bbs_p1003
  • June 14, 2010 Digested PCR product and pSB1C3 with NotI, ligated
  • June 16, 2010 Transformed from ligation
  • June 17, 2010 Success! We did manage to get colonies that grew on Kan/Chlor plates
  • 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

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
  • June 22,2010 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.
  • June 23,2010 Got colonies on plates from the transformations. Set up overnight liquid cultures of 8 white colonies from each ligation. (red colonies are psb1C3)
  • June 24,2010 Minipreped the liquids set up the night before. Digested each miniprep with XbaI and PstI. Also digested each miniprep with just Pst.
  • June 25, 2010 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.
  • June 28 2010 The correct minipreps were nanodropped and the two with the highest concentrations were kept.

Base Plasmid Version 1.2 (Abandoned)

Base Plasmid Version 1.3 (Abandoned)

Base Plasmid Version 2

Selecting Colonies with our Plasmid

Base Plasmid Version 2.1