Team:ETH Zurich/Biology/Cloning

Protocols

[http://www.neb.com/nebecomm/DoubleDigestCalculator.asp Double digest] for cloning

Ligation

1. 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

2. Let the mixture stay for 1h at room temperature
3. Denature the ligase at 65 °C for 5 min

Transformation

1. Thaw competent cells on ice.
2. Add 200 µl cells to 10 µl ligation mix
3. Place the mixture put them on ice for 30 min
4. Heatshock 30 sec at 42 °C
5. Place the mixture again on ice for 2 min
6. Add 4 x SOC medium (800 µl)
7. Let them grow for 60 min at 37 °C
8. Spin cells down for 5 min at low speed
9. Remove the supernatant, resuspend the cells
10. 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
• 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

1. Assemble SDS-PAGE apparatus
2. Load 5 µl protein marker into the first well
3. Load 10 µl of your samples into the following wells (non-induced, induced…)
4. Connect your gel chamber to the power supply, run the gel with 120 V fixed for around 40 min, watch the advance of the blue front
5. When the blue front is ca. 0.5 cm before the end of the gel, stop the run and remove the gels from the apparatus
6. Remove the gel from the glass-plates
7. Add ca. 100 ml Coomassie staining solution to the gel and incubate on the shaker for ca. 30 min
8. Rinse the gel with ddH₂O
9. 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

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