Team:ETH Zurich/Biology/Cloning
From 2011.igem.org
(Difference between revisions)
(→Competent JM 101 cells) |
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Line 153: | Line 153: | ||
Total 50 ml | Total 50 ml | ||
- | '''1 M CaCl<sub>2</sub>''' | + | '''1 M CaCl<sub>2</sub>•2H<sub>2</sub>O''' |
* 7.35 g CaCl<sub>2</sub> | * 7.35 g CaCl<sub>2</sub> | ||
Total 50 ml | Total 50 ml | ||
Line 163: | Line 163: | ||
'''TFBI''' | '''TFBI''' | ||
* 1.2 g RbCl | * 1.2 g RbCl | ||
- | * 0.99 g MnCl<sub>2</sub> | + | * 0.99 g MnCl<sub>2</sub>•4H<sub>2</sub>O |
* 1 ml 1 M CaCl<sub>2</sub> | * 1 ml 1 M CaCl<sub>2</sub> | ||
* 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 sterile | Total 100 ml; bring to pH 5.8 with 0.2 N acetic acid; filter sterile | ||
Line 183: | Line 183: | ||
* aliquote suspension into pre-cooled tubes, 200 µl each | * aliquote 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 | ||
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==Competent DH5α cells== | ==Competent DH5α cells== |
Revision as of 21:02, 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•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 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)