Team:ETH Zurich/Biology/Cloning
From 2011.igem.org
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==SDS page== | ==SDS page== | ||
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+ | '''Cast gels''' | ||
+ | [[File:SDS.png|800px]] | ||
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+ | # Clean glass plates with 30% EtOH (front- and back-glass plate, 10-spike comb) | ||
+ | # Assemble them in SDS-PAGE gel casting apparatus | ||
+ | # Mix the ingredients for the running gel except for APS and TEMED | ||
+ | # Add APS and TEMED to the lower gels solution, mix by gently | ||
+ | # Pour the gels quickly with a 2 mL pipette. Leave about 1 cm below the bottom of the comb for the stacking gel and avoid formation of air-bubbles. | ||
+ | # Overlay your gels very carefully with isopropanol. The isopropanol helps to avoid drying-out of gels during polymerization and puts weight on the gel thereby helping to obtain a smooth surface. | ||
+ | # Wait until all gels have polymerized completely | ||
+ | # Mix the components of the stacking gel while the running gel is polymerizing, leave | ||
+ | out the APS and TEMED | ||
+ | # Remove the isopropanol completely, wash with 0.5 mL of H2O to get rid of all isopropanol | ||
+ | # Add APS and TEMED to the stacking gel solution, mix by gently shaking the Falcon tube | ||
+ | # Pour the stacking gel on top of the running gel | ||
+ | # Insert the comb without introducing air-bubbles. | ||
+ | # Wait until gels have completely polymerized (approx. 30 min) | ||
+ | # Remove the gels from the casting apparatus, remove the combs, and clean the gels from gel-remnants | ||
+ | |||
+ | '''Preparation''' | ||
# Assemble SDS-PAGE apparatus | # Assemble SDS-PAGE apparatus | ||
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# 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 | ||
# Rinse the gel with ddH<sub>2</sub>O | # Rinse the gel with ddH<sub>2</sub>O | ||
- | # 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 12:03, 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
out the APS and TEMED
Preparation
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