Team:ETH Zurich/Biology/Cloning
From 2011.igem.org
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Done according to the [http://openwetware.org/wiki/Preparing_chemically_competent_cells_%28Inoue%29 Inoue protocol] on OpenWetWare | Done according to the [http://openwetware.org/wiki/Preparing_chemically_competent_cells_%28Inoue%29 Inoue protocol] on OpenWetWare | ||
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==Diffusion test in tubes== | ==Diffusion test in tubes== |
Revision as of 01:49, 22 September 2011
Material and methods |
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All protocols, plasmids and primers can be found here. |
Protocols[http://www.neb.com/nebecomm/DoubleDigestCalculator.asp Double digest] for cloning
Ligation
Transformation
PCRPCR mixture
adjust to 50 µl with ddH2O PCR procedure
Colony PCRpick a colony and resuspend in 10 µl ddH2O PCR mixture
adjust to 10 µl with ddH2O PCR procedure
Preparation of glycerol stocksFrom the overnight cultures 15 % glycerol were made and stored at -20 °C AlcR testing
Chemically competent E. coli (JM 101)1 M MOPS
Total 50 ml 1 M CaCl2•2H2O
Total 50 ml 3 M KOAc
Total 50 ml TFBI
Total 100 ml; bring to pH 5.8 with 0.2 N acetic acid; filter sterilize TFBII
filter sterilize Preparation
Chemically competent E. coli (DH5α)Done according to the [http://openwetware.org/wiki/Preparing_chemically_competent_cells_%28Inoue%29 Inoue protocol] on OpenWetWare Diffusion test in tubes
MediumsSOB
adjust to pH 7.5 by adding 1M NaOH SOC
LB medium
Total 1 l M9 minimal medium 10x M9
Total 100 ml autoclave seperatly
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