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Team:Lethbridge/Notebook/Lab Work/Group2
From 2011.igem.org
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Ryan vacationing in Whitefish. Having an awesome time. <br> | Ryan vacationing in Whitefish. Having an awesome time. <br> | ||
Week 4 May 23 - 29 2011 <br> | Week 4 May 23 - 29 2011 <br> | ||
| - | First week Ryan begins working full time in the lab. Successfully used PCR to alter prefix and suffix regions to to the Silver standard for the genes xylE and mms6. That way we can fuse signal sequences. On an agarose gel the DNA strands are of expected size for both xylE and mms6 with the fusion standard. After attempting to cut PCR products of xylE and mms6 and cloning into psb1c3 using red/white screening, no white colonies test positive for the product when the plasmids are restricted and resolved on a gel.<br> | + | First week Ryan begins working full time in the lab. Successfully used PCR to alter prefix and suffix regions to to the Silver standard for the genes xylE and mms6. That way we can fuse signal sequences. On an agarose gel the DNA strands are of expected size for both xylE and mms6 with the fusion standard. After attempting to cut PCR products of xylE and mms6 and cloning into psb1c3 using red/white screening, no white colonies test positive for the product when the plasmids are restricted and resolved on a gel. Miscellaneous plasmids are taken out of the glycerol stock and grown in LB cultures. <br> |
Week 5 May 30 - June 5 2011 <br> | Week 5 May 30 - June 5 2011 <br> | ||
The problem with the ligation of PCR products into psb1c3 is diagnosed as a result of prefix and suffix restriction sites being too close to the ends of the PCR products. Instead of using red/white colony screening and psb1c3 the polymerase used in the PCR produces blunt ends and therefore blunt end ligation is used for cloning with puc19. Different conditions are used in the blunt end ligation. Since puc19 has to be cut bluntly half the samples have the blunt end restriction enzyme heat killed. Blue/white colony screening has to be used in for puc19 cloning which requires X-Gal and IPTG. Solutions of these are made for the plates. Attempts to assemble miscellaneous parts from synthesis vectors to psb1c3 results in positive testing white colonies on an agarose gel for k331025, an enhanced cyan fluorescent protein with a ribosomal binding site and a c-terminally fused arginine residue tail.<br> | The problem with the ligation of PCR products into psb1c3 is diagnosed as a result of prefix and suffix restriction sites being too close to the ends of the PCR products. Instead of using red/white colony screening and psb1c3 the polymerase used in the PCR produces blunt ends and therefore blunt end ligation is used for cloning with puc19. Different conditions are used in the blunt end ligation. Since puc19 has to be cut bluntly half the samples have the blunt end restriction enzyme heat killed. Blue/white colony screening has to be used in for puc19 cloning which requires X-Gal and IPTG. Solutions of these are made for the plates. Attempts to assemble miscellaneous parts from synthesis vectors to psb1c3 results in positive testing white colonies on an agarose gel for k331025, an enhanced cyan fluorescent protein with a ribosomal binding site and a c-terminally fused arginine residue tail.<br> | ||
Week 5 May 30 - June 5 2011 <br> | Week 5 May 30 - June 5 2011 <br> | ||
| + | One colony for the blunt end ligation cloning of an xylE PCR product produces a band at the expected size when the plasmid is restricted and run on a gel. A glycerol stock of this<br> | ||
Week 6 June 6 - 12 <br> | Week 6 June 6 - 12 <br> | ||
Week 7 June 13 - 19 <br> | Week 7 June 13 - 19 <br> | ||
Revision as of 02:19, 15 September 2011
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