Team:ETH Zurich/Biology/Cloning
From 2011.igem.org
(→AlcR testing) |
(→Competent JM 101 cells) |
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* Fluorescence of GFP was measured after 1 h, 2 h and over night, all measurements were done in triplicates | * Fluorescence of GFP was measured after 1 h, 2 h and over night, all measurements were done in triplicates | ||
- | == | + | ==Chemically competent ''e.coli'' (JM 101)== |
'''1 M MOPS''' | '''1 M MOPS''' | ||
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* 1 ml 3 M KOAc | * 1 ml 3 M KOAc | ||
* 15.33 ml glycerol 100 % | * 15.33 ml glycerol 100 % | ||
- | Total 100 ml; bring to pH 5.8 with 0.2 N acetic acid; filter | + | Total 100 ml; bring to pH 5.8 with 0.2 N acetic acid; filter sterilize |
'''TFBII''' | '''TFBII''' | ||
- | * 0.048 g | + | * 0.048 g RbCl |
* 3 ml 1 M CaCl<sub>2</sub> | * 3 ml 1 M CaCl<sub>2</sub> | ||
* 0.4 ml 1 M MOPS pH 7 | * 0.4 ml 1 M MOPS pH 7 | ||
* 7 ml glycerol 100 % | * 7 ml glycerol 100 % | ||
- | filter | + | filter sterilize |
'''Preparation''' | '''Preparation''' | ||
* add 0.5 ml fresh O/N culture to 250 ml LB medium | * add 0.5 ml fresh O/N culture to 250 ml LB medium | ||
- | * | + | * shake at 37 °C until OD<sub>600</sub> is 0.5-0.7 |
- | * cool on ice, spin down at 4 °C/ 3000rpm/ 5 min, put pellet | + | * cool on ice, spin down at 4 °C/ 3000rpm/ 5 min, put pellet on ice |
* resuspend pellet in 5 ml TFBI with pipet, add 70 ml cool TFBI, incubate on ice for 2-4 h | * resuspend pellet in 5 ml TFBI with pipet, add 70 ml cool TFBI, incubate on ice for 2-4 h | ||
- | * | + | * aliquot suspension into pre-cooled tubes, 200 µl each |
* freeze aliquots immediately in dry ice, store at -80 °C | * freeze aliquots immediately in dry ice, store at -80 °C | ||
Revision as of 23:09, 19 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
Competent DH5α cellsDone according the [http://openwetware.org/wiki/Preparing_chemically_competent_cells_%28Inoue%29 protocol] of OpenWetWare Antibioticsampicillin kanamycin chloramphenicol 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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Plasmid listBackbones Plasmid for test system Plasmids for part characterization Plasmid for final system |
Primer list |
Synthesized parts
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