Team:FAU Erlangen/Experiments
Experiments
Medium and buffer production
LB medium (1l liquid)
- 10 g tryptone
- 10 g NaCl
- 5 g yeast extract
- Water
LB medium (1l solid)
- 10 g tryptone
- 10 g NaCl
- 5 g yeast extract
- Water
- 12 g agar
> For selective medium, supplement
with antibiotic as appropiate
BHI medium (1l solid)
- 25 g brain heart infusion
- 12 g agar
PYE medium (1l liquid)
- 2 g peptone
- 1 g yeast extract
- 0.02g CaCl2 * 2H2O
> adjust pH 7.0
PYE medium (1l solid)
- 2 g peptone
- 1 g yeast extract
- 0.02g CaCl2 * 2H2O
- 12 g agar
> adjust pH 7.0
CgXII medium (1l liquid)
- 42 g MOPS
- 20 g (NH4)2SO4
- 1 g KH2PO4
- 1g H2HPO4
> adjust pH 7.0
HEPES Buffer (100ml, 100mM)
- 2.33 g HEPES
> Adjust pH to 7.2 by using NaOH pellets (~1) or adjust pH 2.0 by using HCl
> filter sterile
Western transfer buffer (1l)
- 100 ml 10x transfer buffer
- 200 ml methanol (99.99%)
- VE water
Western blocking buffer
- 5 % Skimmed milk powder in TBST buffer
Schrägger-buffer (500ml)
- 181.71 g 3M TRIS
- 38 ml 1 M HCl
- 7.5 ml 20% SDS
- VE water
Experiments
PCR
PCR program
qPCR:
• Pick colonies with pipette tip and re-suspend in the PCR approach
• Protocol see PCR
Gel electrophoresis
1. Use 1% of agarose diluted in 1x TAE buffer storage in 65°C incubator
2. Pour the solution into the bed, clear all its bubbles with a tip and let it polymerize.
3. Load gel with DNA ladder and DNA sample and fill the gel chamber with 1x TAE buffer
4. Run at 120V for 45 minutes
5. Incubate the gel in EtBr for 10 minutes and wash with water
6. Image gel under UV light
Transformation with DH5α competent cells
1. Throw competent cells on ice.
2. Add 2µl of the plasmid to the competent cells
3. Place the mixture on ice for 15 minutes. Do not mix.
4. Heat shock at 42°C for 30 seconds. Do not mix.
5. Transfer tubes to ice for 2 minutes.
6. Add 950µl of room temperature SOC-media to the tube.
7. Incubate the tube at 37°C for 60 minutes 250 rpm.
8. Warm selection plates to 37°C
9. Centrifuge and spread 100µl of the cells onto the selection plates with antibiotics
10. Incubate Overnight at 37°C
SDS PAGE
Western Blot
1. Load SDS gels + prepare samples
• Put gels in overflow chamber and fill between the gels with 1xCathode buffer and on the outside with 1xAnode buffer
• Remove comb and apply prepared samples
• Loading of gels with glass syringe
Prepare Samples:
• Transfer each sample with loading dye = Lämmli buffer 1:1 (2x PAP)
• Incubate 5min at 100°C
• ~5µl, protein marker = Page Ruler Prestained
> ~1.5h at 120V
2. Blotting
• Adjust filter paper (=Whatmanpaper) and PVDF Membrane
• Soak PVDF membrane for 5min in 60% Methanol
• Wash PVDF membrane with water
• Wash membrane 5min in 1x Western Transfer buffer
• Soak filter paper in 1x Transfer buffer
• Building of gel”sandwich“ and removing of bubbles
o For 1h at 120mA (2 gels: 230mA, 3 gels: 300mA)
• Discard filter paper, perhaps coloring of gel
• Wash membrane 3x 15min in 1x TBST
3. Blocking
• Incubate membrane 1h in Blocking buffer at 4°C (= 5% Skimmed milk powder in TBST, 5g Skimmed milk powder in 100ml TBST)
4. Incubation
• Incubate membrane with primary AK o/n at 4°C
o Normally: 1:5000 (in blocking solution)
o This time: 1:3000 (10ml +4μl α-His)
• Wash membrane 4x 15min with TBST
• Shake membrane with secondary AK (1:10000, 20ml blocking buffer + 2μl AK) for 1h at 4°C
• Wash membrane 4x 15min with TBST
5. Detecion
• Prepare fresh: 5ml incubation buffer with 33μl NBT = nitro blue tetrazolium (50mg/50ml) and 16.5μl BCIP = 5-bromo-4-chloro-3-indolphsophatase (50mg/50ml) -> 25mg/ml = 33μl BCIP
• Insert ~3ml one after another until membrane is fully covered
• Incubate 1min-1h in the dark until coloring is sufficient (~30min)
• Stop reaction with water
• Dry membrane
Plasmid extraction mini-prep.
• NucleoSpin-Kit (according to kit protocol)
Plasmid extraction midi- prep.
• Xtra Midi-Kit (according to manufacturer instructions)
Preparaton of chromosomal DNA (Corynebacterium)
Prepare an overnight culturein 20-30 ml BHI at 30°C
• 4500 g at 15min
• Discard supernatant
• Resuspend pellet in 3 ml TE with pH 8 (1.5ml falcon)
• Add a spatula tip-full of lysozyme + 30 µl Rnaset (10µg/µl)
• Incubate at 37°C, 125 rpm shaker, 2h
• Add 210 µl 20% SDS (respectively 420 µl 10% SDS) + 30 µl proteinkinase K (20 µg/ml) (or 60 µl proteinkinase (10µg/ml))
• Invert sample
• Incubate for 2h in dryer at 65 °C
• Add 600 µl 5 M NaCl
• 30 min in dryer at 64°C
Next steps must be developed in a fume hood
• 4.5 ml chloroform: phenol: isoamylalkohol (25: 24: 1)
• Centrifuge for 15 min, 8900g, 4°C
• Upper phase into a new falcon with 2ml isopropanol
• Invert many times until you see a cloud
• Fish the DNA – into prepared eppis with 500 µl 70% EtOH
• Centriuge 2x 10 min 500 µl EtOH (70%)
• Dry in speedvac
• Resuspend in 100 µl TE pH 8 (overnight, RT)
Isolations
RsaA (p3, C. crescentus)
• oD600 = 0.53
• 6x 6ml 10min 10000g
• Dilute HEPES Puffer 1/10 (= 0.01M)
• Wash 2x in 10mM HEPES pH 7.2; 10000g 10min
• Resuspend In 200μl 10mM HEPES pH 2.0
• Incubate 10min RT (20°C)
• Transfer to 1.5ml Eppis (3x 200μl each)
• 10min at 13000rpm
• supernatant in new 1.5ml Eppi
• Adjust pH to 7.0 with 1M NaOH
PS2 isolation (p2, C. glutamicum)
• Overnigth culture OD600= 3.03
• 6 falcons with each 30ml culture (OD=1.0)
• Centrifugate 5 minutes 5000g at room temperature
• Discard supernatant
• Resuspend pellet in 1.5ml 50mM Tris pH 6.8 or 50mM HEPES buffer pH 6.8
• Incubate 1 hour at room temperature
• Centrifugate 5 minutes 5000g at room temperature
• Transfer supernatant in a new eppi and storage on ice
SbsB isolation (p1, E. coli)
• Overnight culture for first purification: inoculates 100ml LB + Amp (100 μl) + E. coli (containing SbsB plasmid) overnight (37°C)
• Overexpression for first purification:
1L LB + 20ml overnight culture + 1ml Amp. Let it grow for 1.5h (oD 1.35) and induce with 1ml 1MIPTG for 4h. Centrifuge 10min/ 3000g/ 4°C and store pellet overnight at -20°C.
• Lysis for purification: resuspend the bacteria pellet in 8ml binding buffer (Puffer A with protease inhibiter (1 pill). Add 30ml of the Lysozyme solution. Sonicate 30x 9 circles. 5 times with 5min pause in between always keep on ice, with big sonde, 50% power. Centrifuge 30min at 8500rpm 4°C 1h 30min, add 30 ml DNase I and incubate for 30 min.
• Protein purification with Ni – NTA: prepare columns: 4ml Ni-NTA per column (2 columns) in 4°C Room and discard supernatant from the columns. Wash Ni-NTA slurry in columns three times with binding buffer (Puffer A). Apply lysate supernatant to columns and incubate at 4°C for around 2h/over nigh.t
• Equilibrate column with buffer A. Fill column with buffer a and let it flow through, (flowthrough was stored for analyzation). Protein elutes with elution buffer: gradient: 25%, 50%, 75%, 9x 100% (9 fractions per column).
• Check with sds gel.
Deviations from the experiments: see notebook