Team:Lethbridge/Notebook/Lab Work/Group2
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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. | + | 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> | ||
+ | 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. Attempts to move the remaining miscellaneous parts into psb1c3 produce no positive testing white colonies. <br> | ||
Week 6 June 6 - 12 <br> | Week 6 June 6 - 12 <br> | ||
- | <br> | + | A glycerol stock of the one colony of xylE and k331025 were made after they were re-transformed and grown in liquid LB culture. After another attempt the mms6 PCR product still refuses to be cloned via blunt end ligation. The same can be said for the another assembly of the miscellaneous parts to psb1c3. <br> |
Week 7 June 13 - 19 <br> | Week 7 June 13 - 19 <br> | ||
+ | <br> | ||
Week 8 June 20 - 26 <br> | Week 8 June 20 - 26 <br> | ||
Week 9 June 27 - July 3 <br> | Week 9 June 27 - July 3 <br> |
Revision as of 03:40, 15 September 2011
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