Team:ETH Zurich/Biology/Cloning
From 2011.igem.org
(Difference between revisions)
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- | {{:Team:ETH Zurich/Templates/Section|SectionNum=2|SectionName= | + | {{:Team:ETH Zurich/Templates/Section|SectionNum=2|SectionName=Double Digest for cloning|SectionType==|Content= |
{| border="1" align="center" style="text-align:left;" | {| border="1" align="center" style="text-align:left;" | ||
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# 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= | + | {{:Team:ETH Zurich/Templates/Section|SectionNum=4|SectionName=PCR|SectionType==|Content= |
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'''PCR mixture''' | '''PCR mixture''' | ||
* 10 µl 10x Phusion buffer | * 10 µl 10x Phusion buffer | ||
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* storage at 4°C | * storage at 4°C | ||
}} | }} | ||
- | {{:Team:ETH Zurich/Templates/Section|SectionNum=5|SectionName= | + | {{:Team:ETH Zurich/Templates/Section|SectionNum=5|SectionName=Colony PCR|SectionType==|Content= |
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pick a colony and resuspend in 10 µl ddH<sub>2</sub>O | pick a colony and resuspend in 10 µl ddH<sub>2</sub>O | ||
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* storage at 4 °C | * storage at 4 °C | ||
}} | }} | ||
- | == Preparation of glycerol stocks== | + | |
+ | {{:Team:ETH Zurich/Templates/Section|SectionNum=6|SectionName=Preparation of glycerol stocks|SectionType==|Content= | ||
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 | ||
- | + | }} | |
- | ==AlcR testing== | + | {{:Team:ETH Zurich/Templates/Section|SectionNum=7|SectionName=AlcR testing|SectionType==|Content= |
* preparatory overnight culture of JM101 with AlcR-testsystem in LB medium | * preparatory overnight culture of JM101 with AlcR-testsystem in LB medium | ||
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* Cells were incubated at 37 °C in closed tubes | * Cells were incubated at 37 °C in closed tubes | ||
* 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)== | + | {{:Team:ETH Zurich/Templates/Section|SectionNum=8|SectionName=Chemically competent ''E. coli'' (JM 101)|SectionType==|Content= |
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'''1 M MOPS''' | '''1 M MOPS''' | ||
* 10.46 g MOPS | * 10.46 g MOPS | ||
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* aliquot suspension into pre-cooled tubes, 200 µl each | * 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 | ||
- | + | }} | |
- | ==Chemically competent ''E. coli'' (DH5α)== | + | {{:Team:ETH Zurich/Templates/Section|SectionNum=9|SectionName=Chemically competent ''E. coli'' (DH5α)|SectionType==|Content= |
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Done according to the [http://openwetware.org/wiki/Preparing_chemically_competent_cells_%28Inoue%29 Inoue protocol] on OpenWetWare | Done according to the [http://openwetware.org/wiki/Preparing_chemically_competent_cells_%28Inoue%29 Inoue protocol] on OpenWetWare | ||
- | + | }} | |
- | ==SDS page== | + | {{:Team:ETH Zurich/Templates/Section|SectionNum=10|SectionName=SDS page|SectionType==|Content= |
(D-BSSE MolBioSkript 2011) | (D-BSSE MolBioSkript 2011) | ||
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# 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 are clearly visible and background signals are almost completely disappeared | # 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 | ||
+ | }} | ||
+ | {{:Team:ETH Zurich/Templates/Section|SectionNum=11|SectionName=Western Blot|SectionType==|Content= | ||
+ | }} | ||
- | = | + | {{:Team:ETH Zurich/Templates/Section|SectionNum=12|SectionName=Diffusion test in tubes|SectionType==|Content= |
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* agarose with ''E. coli'' with IPTG inducible GFP were filled in different tubes | * agarose with ''E. coli'' with IPTG inducible GFP were filled in different tubes | ||
* on end of the tube was put in a IPTG solution | * on end of the tube was put in a IPTG solution | ||
+ | }} | ||
- | ==Mediums== | + | {{:Team:ETH Zurich/Templates/Section|SectionNum=13|SectionName=Mediums|SectionType==|Content= |
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'''Agar plates''' | '''Agar plates''' | ||
* 18g/l Agar added per liter respective medium | * 18g/l Agar added per liter respective medium | ||
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* 1 % (w/v) thiamine solution | * 1 % (w/v) thiamine solution | ||
* 20 % (w/v) glucose | * 20 % (w/v) glucose | ||
- | + | }} | |
- | + | {{:Team:ETH Zurich/Templates/Section|SectionNum=14|SectionName=Antibiotics|SectionType==|Content= | |
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* Ampicilin: 50ug/ml for low copy and 150ug/ml for high copy plasmids | * 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 | * 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 | * Kanamycin: 10ug/ml for low copy and 30ug/ml for high copy plasmids | ||
+ | }} |
Revision as of 19:43, 24 October 2011
Material and methods |
| |
All protocols, plasmids and primers can be found here. |
Double Digest for cloning
{
Ligation
- Ligation mix
- 1:5 ratio of plasmid backbone : Insert
- 1 µl 10 x T4 Ligase buffer (vortex)
- 0.5 µl T4 Ligase
- adjust to 10 µl with ddH2O
- Let the mixture stay for 1h at room temperature
- Denature the ligase at 65 °C for 5 min
Acetaldehyde Sensor
Transformation
- Thaw competent cells on ice.
- Add 200 µl cells to 10 µl ligation mix
- Place the mixture put them on ice for 30 min
- Heatshock 30 sec at 42 °C
- Place the mixture again on ice for 2 min
- Add 4 x SOC medium (800 µl)
- Let them grow for 60 min at 37 °C
- Spin cells down for 5 min at low speed
- Remove the supernatant, resuspend the cells
- Spread 100 µl of the cells onto the plates.
PCR
PCR mixture
- 10 µl 10x Phusion buffer
- 1 µl 10 mM dNTPs
- 2.5 µl 10 µM forward primer
- 2.5 µl 10 µM reverse primer
- 1 ng DNA template
- 0.2 µl Phusion polymerase
- optional DMSO
adjust to 50 µl with ddH2O
PCR procedure
- initial denaturation: 98 °C for 30 sec
- 35 cycles
- denaturation: 98 °C for 5 sec
- annealing: annealing temperature (see Primer list) for 10 sec
- extension: 72 °C for 30 sec per kb
- final extension: 72°C for 5 min
- storage at 4°C
Colony PCR
pick a colony and resuspend in 10 µl ddH2O
PCR mixture
- 1 µl colony
- 1 µl 10x taq buffer
- 0.8 µl dNTPs
- 0.05 µl 10 µM forward primer
- 0.05 µl 10 µM reverse primer
- 0.1 µl Taq
adjust to 10 µl with ddH2O
PCR procedure
- initial denaturation: 95 °C for 30 sec
- 25 cycles
- denaturation: 95 °C for 30 sec
- annealing: 55 °C for 30 sec
- extension: 68 °C for 90 sec
- final extension: 68 °C for 5 min
- storage at 4 °C
Preparation of glycerol stocks
From the overnight cultures 15 % glycerol were made and stored at -20 °C
AlcR testing
- preparatory overnight culture of JM101 with AlcR-testsystem in LB medium
- cultures in M9 minimal medium
- all final experiments were done in 15 ml falcons with 3 ml cells
- AlcR production was induced with either 0, 1, 100 ng/ml anhydrotetracycline added at 4 °C
- For AlcR activation 0, 10, 1000 µM acetaldehyde was added at 4 °C
- Cells were incubated at 37 °C in closed tubes
- 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
- 10.46 g MOPS
Total 50 ml
1 M CaCl2•2H2O
- 7.35 g CaCl2
Total 50 ml
3 M KOAc
- 14.72 g KOAc
Total 50 ml
TFBI
- 1.2 g RbCl
- 0.99 g MnCl2•4H2O
- 1 ml 1 M CaCl2
- 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 sterilize
TFBII
- 0.048 g RbCl
- 3 ml 1 M CaCl2
- 0.4 ml 1 M MOPS pH 7
- 7 ml glycerol 100 %
filter sterilize
Preparation
- add 0.5 ml fresh O/N culture to 250 ml LB medium
- shake at 37 °C until OD600 is 0.5-0.7
- 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
- spin down at 4 °C/3krpm/ 5min
- resuspend pellet in 10 ml TFBII
- aliquot suspension into pre-cooled tubes, 200 µl each
- freeze aliquots immediately in dry ice, store at -80 °C
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
{
Western Blot
Diffusion test in tubes
- agarose with E. coli with IPTG inducible GFP were filled in different tubes
- on end of the tube was put in a IPTG solution
Mediums
Agar plates
- 18g/l Agar added per liter respective medium
SOB
- 0.5 % (w/v) yeast extract
- 2 % (w/v) tryptone
- 10 mM NaCl
- 2.5 mM KCl
- 20 mM MgSO4
adjust to pH 7.5 by adding 1M NaOH
SOC
- SOB
- 20 mM glucose
LB medium
- 10 g Bacto-tryptone
- 5 g yeast extract
- 10 g NaCl
Total 1 l
M9 minimal medium
10x M9
- 12.8 g Na2HPO4
- 3 g KH2PO4
- 0.5 g NaCl
- 1 g NH4Cl
Total 100 ml
autoclave seperatly
- 10 x M9
- 1 M MgSO4
- 1 M CaCl2
- 1 % (w/v) thiamine solution
- 20 % (w/v) glucose
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