Team:ETH Zurich/Biology/Cloning
From 2011.igem.org
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# Load 10 µl of your samples into the following wells (non-induced, induced…) | # Load 10 µl of your samples into the following wells (non-induced, induced…) | ||
# Connect your gel chamber to the power supply, run the gel with 120 V fixed for around 40 min, watch the advance of the blue front | # Connect your gel chamber to the power supply, run the gel with 120 V fixed for around 40 min, watch the advance of the blue front | ||
- | # When the blue front is ca. 0.5 cm before the end of the gel, stop the run and | + | # When the blue front is ca. 0.5 cm before the end of the gel, stop the run and remove the gels from the apparatus |
- | remove the gels from the apparatus | + | |
# Remove the gel from the glass-plates | # Remove the gel from the glass-plates | ||
# Add ca. 100 ml Coomassie staining solution to the gel and incubate on the shaker for ca. 30 min | # Add ca. 100 ml Coomassie staining solution to the gel and incubate on the shaker for ca. 30 min | ||
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# Add ca. 100 ml destaining solution to the gel, incubate for until the protein-bands | # Add ca. 100 ml destaining solution to the gel, incubate for until the protein-bands | ||
are clearly visible and background signals are almost completely disappeared | are clearly visible and background signals are almost completely disappeared | ||
+ | |||
==Western Blot== | ==Western Blot== | ||
Revision as of 11:54, 13 October 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 SDS page
are clearly visible and background signals are almost completely disappeared Western BlotDiffusion test in tubes
MediumsAgar plates
SOB
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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Antibiotics
- Ampicilin: 50ug/ml for low copy and 150ug/ml for high copy plasmids
- Chloramphenicol: 34ug/ml for low copy and 100ug/ml for high copy plasmids
- Kanamycin: 10ug/ml for low copy and 30ug/ml for high copy plasmids