Team:ETH Zurich/Biology/Cloning
From 2011.igem.org
(→Chemically competent E. coli (JM 101)) |
|||
Line 15: | Line 15: | ||
== '''Protocols''' == | == '''Protocols''' == | ||
- | + | {{:Team:ETH Zurich/Templates/Section|SectionNum=2|SectionName=[http://www.neb.com/nebecomm/DoubleDigestCalculator.asp Double digest] for cloning|SectionType==|Content= | |
- | == [http://www.neb.com/nebecomm/DoubleDigestCalculator.asp Double digest] for cloning == | + | |
- | + | ||
{| border="1" align="center" style="text-align:left;" | {| border="1" align="center" style="text-align:left;" | ||
| | | | ||
Line 56: | Line 54: | ||
* adjust to 10 µl ddH<sub>2</sub>O | * adjust to 10 µl ddH<sub>2</sub>O | ||
|} | |} | ||
- | |||
<center> ⇒ incubate for 2 h at 37°C and gel purify </center> | <center> ⇒ incubate for 2 h at 37°C and gel purify </center> | ||
+ | }} | ||
- | == Ligation == | + | {{:Team:ETH Zurich/Templates/Section|SectionNum=2|SectionName=Ligation|SectionType==|Content= |
- | + | ||
# Ligation mix | # Ligation mix | ||
#* 1:5 ratio of plasmid backbone : Insert | #* 1:5 ratio of plasmid backbone : Insert | ||
Line 70: | Line 67: | ||
# Denature the ligase at 65 °C for 5 min | # Denature the ligase at 65 °C for 5 min | ||
+ | {{:Team:ETH Zurich/Templates/Section|SectionNum=3|SectionName=Acetaldehyde Sensor|SectionType==|Content= | ||
== Transformation == | == Transformation == | ||
Line 83: | Line 81: | ||
# Spread 100 µl of the cells onto the plates. | # Spread 100 µl of the cells onto the plates. | ||
+ | {{:Team:ETH Zurich/Templates/Section|SectionNum=4|SectionName=Acetaldehyde Sensor|SectionType==|Content= | ||
== PCR == | == PCR == | ||
Line 104: | Line 103: | ||
* final extension: 72°C for 5 min | * final extension: 72°C for 5 min | ||
* storage at 4°C | * storage at 4°C | ||
- | + | }} | |
+ | {{:Team:ETH Zurich/Templates/Section|SectionNum=5|SectionName=Acetaldehyde Sensor|SectionType==|Content= | ||
== Colony PCR == | == Colony PCR == | ||
Line 126: | Line 126: | ||
* final extension: 68 °C for 5 min | * final extension: 68 °C for 5 min | ||
* storage at 4 °C | * storage at 4 °C | ||
- | + | }} | |
== Preparation of glycerol stocks== | == Preparation of glycerol stocks== | ||
From the overnight cultures 15 % glycerol were made and stored at -20 °C | From the overnight cultures 15 % glycerol were made and stored at -20 °C |
Revision as of 19:30, 24 October 2011
Material and methods |
| |
All protocols, plasmids and primers can be found here. |
Protocols
[http://www.neb.com/nebecomm/DoubleDigestCalculator.asp Double digest] for cloning
{
{{:Team:ETH Zurich/Templates/Section|SectionNum=2|SectionName=Ligation|SectionType==|Content=
{{:Team:ETH Zurich/Templates/Section|SectionNum=3|SectionName=Acetaldehyde Sensor|SectionType==|Content= Transformation
Acetaldehyde SensorPCRPCR mixture
adjust to 50 µl with ddH2O PCR procedure
Acetaldehyde SensorColony 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(D-BSSE MolBioSkript 2011) Cast gels
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
|
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