From 2011.igem.org
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| |style="border-left: none;"|[[#Protocols|Protocols]] | | |style="border-left: none;"|[[#Protocols|Protocols]] |
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- | |[[#Primer list|Primer list]]
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| * 1 % (w/v) thiamine solution | | * 1 % (w/v) thiamine solution |
| * 20 % (w/v) glucose | | * 20 % (w/v) glucose |
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- | == '''Plasmid list''' ==
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- | '''Backbones'''
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- | '''Plasmids for test system'''
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- | '''Plasmids for part characterizations'''
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- | '''Plasmids for final system'''
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- | == '''Synthesized parts''' ==
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- | * LacI<sub>M1</sub>
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- | * AlcR (codon optimized)
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Revision as of 01:33, 22 September 2011
Material and methods
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All protocols, plasmids and primers can be found here.
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Protocols
[http://www.neb.com/nebecomm/DoubleDigestCalculator.asp Double digest] for cloning
| Variant 1
| Variant 2
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Plasmid
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- x µl Plasmid (~2 µg if possible)
- 2 µl EcoRI
- 2 µl XbaI
- 5 µl buffer 4
- 0.5 µl BSA
- adjust to 10 µl ddH2O
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- x µl Plasmid (~2 µg if possible)
- 3 µl PstI
- 2 µl Spe I
- 5 µl buffer 2
- 0.5 µl BSA
- adjust to 10 µl ddH2O
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Insert
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- x µl Insert (~2 µg if possible)
- 2 µl EcoRI
- 3 µl Spe I
- 5 µl buffer 1
- 0.5 µl BSA
- adjust to 10 µl ddH2O
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- x µl Insert (~2 µg if possible)
- 3 µl XbaI
- 2 µl Pst 1
- 5 µl buffer 3
- 0.5 µl BSA
- adjust to 10 µl ddH2O
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⇒ incubate for 2 h at 37°C and gel purify
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
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
Total 50 ml
1 M CaCl2•2H2O
Total 50 ml
3 M 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
- 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
Antibiotics
- ampicillin
- kanamycin
- chloramphenicol
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
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
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
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