Team:ETH Zurich/Biology/Cloning
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Revision as of 16:55, 19 September 2011 by Sabineoesterle (Talk | contribs)
Material and methods |
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All protocols, plasmids and primers can be found here. |
Protocols
Double digest for cloning
Variant 1 | Variant 2 | |
Plasmid |
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Insert |
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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 10µl liagtion mix
- Place the mixture put them on ice for 30 min
- Heatschock 30 sec at 42 °C
- Place the mixture again on ice for 2 min
- Add 4 x SOC medium (800 µl)
- Let them growth 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: anealingtemperatur (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
Colonie PCR
pick a colonie and resupsend in 10 µl ddH2O
PCR mixture
- 1 µl colony
- 1 µl 10x taq buffer
- 0.8 µl dNTPs
- 0.05 µl forward primer
- 0.05 µl 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
Whole plasmid PCR
Preparation of glycerol stocks
AlcR testing
- preparatory overnight culture of JM101 with AlcR-testsystem in LB medium
- culturs 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 at 4°C
- For AlcR activation 0, 10, 1000 µM acetaldehyde was added at 4°C
- Florescence of GFP was measured after 1 h, 2 h and over night, all measurements were done in triplicates
Antibiotics
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
- 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
Used parts from registry
- BBa R0040 PTet
- BBa R0051 λP
- BBa R0010 Plac
- BBa R0061 Plux
- BBa J23100 Pconst
- BBa C0061 LuxILVA
- BBa C0040 TetRLVA
- BBa C0051 CI
- BBa C0062 LuxR
- BBa B0015 Double Terminator
Plasmid list
Primer list
Synthesized parts
- LacIM1
- AlcR (codon optimized)