IGEM:University of East Anglia (UEA), Norwich, UK/2009/Notebook/NRP-UEA-Norwich iGEM/2013/08/29: Difference between revisions
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==Floor Two== | ==Floor Two== | ||
As the agarose gel from the 22/08 was unsuccessful, the restriction digest of 3b and AntGpneo with PstI and NdeI was repeated. The miniprep BL21 cells that contain pETAntA and AntGpRFP plasmids were digested with XbaI. All samples were analysed by gel electrophoresis. <br> | As the agarose gel from the 22/08 was unsuccessful, the restriction digest of 3b and AntGpneo with PstI and NdeI was repeated. The miniprep BL21 cells that contain pETAntA and AntGpRFP plasmids were digested with XbaI. All samples were analysed by gel electrophoresis. <br> | ||
The gel was successful and a large fragment from 3b and small fragment from AntGpneo were excised, purified and ligated overnight at 4°C. | The gel was successful and a large fragment from 3b and small fragment from AntGpneo were excised, purified and ligated overnight at 4°C. Colonies one and two from the plate transformed with BL21 (pET28AntA + AntGRFP) cells looked unusual on the gel. They appeared to contain unexpected DNA. But Colonies three and four from the same plate looked promising and would need further investigating to establish whether they contain two plasmids. | ||
Two colonies from the plates containing samples 3a and Bba_J04450 were inoculated into 10ml cultures of LB and Chlorophenicol antibiotic. The four cultures (two colonies from each) were left to grow overnight at 37°C. | Two colonies from the plates containing samples 3a and Bba_J04450 were inoculated into 10ml cultures of LB and Chlorophenicol antibiotic. The four cultures (two colonies from each) were left to grow overnight at 37°C. | ||
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Floor OneAgarose gel electrophoresis showed that something had gone wrong during PCR - genomic DNA was visible. Floor TwoAs the agarose gel from the 22/08 was unsuccessful, the restriction digest of 3b and AntGpneo with PstI and NdeI was repeated. The miniprep BL21 cells that contain pETAntA and AntGpRFP plasmids were digested with XbaI. All samples were analysed by gel electrophoresis.
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