Team:ETH Zurich/Biology/Cloning
From 2011.igem.org
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* Cells were incubated at 37 °C in closed tubes | * Cells were incubated at 37 °C in closed tubes | ||
* Florescence of GFP was measured after 1 h, 2 h and over night, all measurements were done in triplicates | * Florescence of GFP was measured after 1 h, 2 h and over night, all measurements were done in triplicates | ||
+ | |||
+ | ==Competent JM 101 cells== | ||
+ | |||
+ | '''1 M MOPS''' | ||
+ | * 10.46 g MOPS | ||
+ | Total 50 ml | ||
+ | |||
+ | '''1 M CaCl<sub>2</sub>''' | ||
+ | * 7.35 g CaCl<sub>2</sub> | ||
+ | Total 50 ml | ||
+ | |||
+ | '''3 M KOAc''' | ||
+ | * 14.72 g KOAc | ||
+ | Total 50 ml | ||
+ | |||
+ | '''TFBI''' | ||
+ | * 1.2 g RbCl | ||
+ | * 0.99 g MnCl<sub>2</sub>*4H<sub>2</sub>O | ||
+ | * 1 ml 1 M CaCl<sub>2</sub> | ||
+ | * 1 ml 3 M KOAc | ||
+ | * 15.33 ml glycerol 100% | ||
+ | Total 100 ml; bring to pH 5.8 with 0.2 N acetic acid; filter sterile | ||
+ | |||
+ | '''TFBII''' | ||
+ | * 0.048 g RBCl | ||
+ | * 3 ml 1 M CaCl<sub>2</sub> | ||
+ | * 0.4 ml 1 M MOPS pH 7 | ||
+ | * 7 ml glycerol 100 % | ||
+ | filter steril | ||
+ | |||
+ | '''Preparation''' | ||
+ | * add 0.5 ml fresh O/N culture to 250 ml LB medium | ||
+ | * Shake at 37 °C untill OD<sub>600</sub> is 0.5-0.7 | ||
+ | * cool on ice, spin down at 4 °C/ 3000rpm/ 5 min, put pellet of ice | ||
+ | * resuspend pellet in 5 ml TFBI with pipet, add 70 ml cool TFBI, incubate on ice for 2-4 h | ||
+ | * aliquote suspension into pre-cooled tubes, 200 µl each | ||
+ | * freeze aliquots immediately in dry ice, store at -80 °C | ||
+ | |||
+ | |||
+ | ==Competent DH5α cells== | ||
+ | |||
+ | Done according the [http://openwetware.org/wiki/Preparing_chemically_competent_cells_%28Inoue%29 protocol] of OpenWetWare | ||
==Antibiotics== | ==Antibiotics== |
Revision as of 20:57, 19 September 2011
Material and methods |
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All protocols, plasmids and primers can be found here. |
ProtocolsDouble digest for cloning
Ligation
Transformation
PCRPCR mixture
adjust to 50 µl with ddH2O PCR procedure
Colonie PCRpick a colonie and resupsend in 10 µl ddH2O PCR mixture
adjust to 10 µl with ddH2O PCR procedure
Whole plasmid PCRPreparation of glycerol stocksFrom the overnight cultures 15 % glycerol were made and storage at -20 °C AlcR testing
Competent JM 101 cells1 M MOPS
Total 50 ml 1 M CaCl2
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 sterile TFBII
filter steril Preparation
Competent DH5α cellsDone according the [http://openwetware.org/wiki/Preparing_chemically_competent_cells_%28Inoue%29 protocol] of OpenWetWare AntibioticsMediumsSOB
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 list |
Primer list |
Synthesized parts
- LacIM1
- AlcR (codon optimized)