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Team:DTU-Denmark/Notebook/19 July 2013
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| - | * colony PCR to verify transformants from [[Team:DTU-Denmark/Notebook/ | + | * colony PCR to verify transformants from [[Team:DTU-Denmark/Notebook/18_July_2013&action=edit| 18-07-2013]] |
* gel electrophoresis to verify insert after colony PCR (AMO, HAO), plasmid isolation | * gel electrophoresis to verify insert after colony PCR (AMO, HAO), plasmid isolation | ||
Revision as of 11:30, 19 July 2013
Contents |
208
Main purpose
- USER reaction and transformation
Who was in the lab
Gosia, Henrike
Procedure
- plasmid isolation
- colony PCR to verify transformants from 18-07-2013
- gel electrophoresis to verify insert after colony PCR (AMO, HAO), plasmid isolation
- restriction analysis of isolated plasmids
Plasmid isolation
According to protocol in QIAprep Spin Miniprep Kit
Colony PCR
Master mix was prepared based on standard colony PCR procedure.
For AMO primers 29a, 29b were used (no USER primers this time) and for HAO primers 28a, 28b were used. We used Phusion polymerase. We obtained 7 transformants after yesterday transformation with AMO in pZA21 and 3 transformants which may contain HAO in pZA21. On negative control plate 3 colonies grew.
PCR parameters according to standard reaction with differences in annealing temperatures (for AMO -> 54°C, for HAO -> 56°C) and with 3 min of extension time. Expected fragments lengths are AMO - 3074 bp, HAO - 2816 bp.
