Team:Bielefeld-Germany/Labjournal/Molecular
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Revision as of 22:57, 29 September 2013
Protocols
Genetic Engineering
Whole Genome Isolation
- For the isolation following kit has been used: Fermentas GeneJET™ Plasmid Miniprep Kit
- Standard Protocol:
- Centrifuge 10 mL of over-night liquid culture
- Resuspend pellet in 800 µL of resuspension solution
- Ribolyse three times for 60 s with 6500 rpm (30 s break between every run, 400 mg beads)
- Centrifuge 3 min at 10,000 rpm
- Transfer 500 µL of supernatant into new 2 mL tube
- Add 500 µL of lysis buffer, invert 4 - 6 times
- Add 700 µL of neutralisation buffer, invert 4 - 6 times
- Centrifuge 10 min at 12,000 rpm
- Transfer 2 x 600 µL of supernatant into spin column (centrifuge down each time for 30 s at 12,000 rpm)
- Two wash steps with 750 µL of washing buffer
- Dry column
- Elute in 75 µL of elution buffer
Generating electrocompetent cells
- Material:
- 550 mL LB-Medium
- 1 L cooled bidest. H2O
- 150 mL cooled 10 % glycerine
- 10 pre-cooled 50 mL Falcons
- Protocol:
- Inoculate 2x3 mL LB with bacterial stock; incubate over night at 37 °C and 200 rpm
- Inoculate 2x250 mL LB with the over night cultures in 1-litre-flask at 37 °C and 140 rpm
- Incubate until OD600 0,4-0,6
- Cool the culture 15-30 minutes on ice
- Onwards all steps at 4°C
- Divide the cultures into cooled 50 mL Falcons and centrifugate at 4000 rpm, 4 °C for 15 minutes, make sure to slowly accelerate and deccelerate
- Discard supernatant
- Resuspend pellet in 5 mL cooled bidest H2O (and don't get frustrated while doing it, keep shaking gently)
- Pool two suspensions each, add bidest H2O up to 50 mL and centrifugate again (see centrifugation above)
- Discard supernatant
- Resuspend pellet in 5 mL cooled bidest H2O
- Add bidest H2O up to 50 mL and centrifugate again (see centrifugation above)
- Discard supernatant
- Resuspend pellet in 5 mL cooled 10 % glycerine
- Transfer suspensions in two 50 mL Falcons and centrifugate again (see centrifugation above)
- Discard supernatant
- Add volume of 10 % glycerine that is approximately equal to the volume of the pellet and resuspend
- Divide cells in 50 μL aliquots and freeze in liquid N2 immediately
- Store at -80 °C
Transformation via electroporation
- Thaw 50 µL competent E.coli cells on ice, dilute with icecold 50 µL glycerine (10 %) if necessary
- Add 0.5-5 µL plasmid to 50 µl electrocompetent cells
- Store cells on ice for 1 minute
- Electroporate at U = 2.5 kV, C = 25 µF, R = 400 Ώ
- Transfer transformation reaction to 450 µL SOC-Medium and shake 1 h at 37 °C
- Centrifuge 2 min at 2000 rpm and plate on selective LB-Medium
- Incubate over night at 37 °C
Analytics
SDS-PAGE
- Pouring the polyacrylamide gel
- Make a master mix for the stacking and separating gel without adding ammonium persulfate and TEMED
- Aliquote 6,5 mL for each separating and 2,5 mL for each stacking gel
- Add ammonium persulfate and TEMED to each separating gel aliquote and pour the solution quickly into your gel casting form. Leave about 2 centimeters below the bottom of the comb for the stacking gel
- Layer isopropanol on top of the gel
- Leave the separating gel at room temperature for >60 minutes to polymerize
- Remove isopropanol and wait until the surface is dry
- Add ammonium persulfate and TEMED to each separating gel aliquote and pour the solution quickly into your gel casting form
- Insert comb without getting bubbles stuck underneath
- Leave the gel at room temperature for >60 minutes to polymerize
- For storage:
- Remove sealing and store the gel wrapped in moistened paper towel at 4°C
- Preparing the sample
- Mix your protein mixture 4:1 with Laemmli-buffer (30 mL protein solution + 10 mL Laemmli-buffer)
- Heat for 5 minutes at 95 °C
- Running the gel
- Remove sealing, put the polymerized gel into gel box and pour SDS running buffer into the negative and positive electrode chamber
- Remove comb without destroying the gel pocket
- Pipet the sample into the gel pockets, adjusting the volume according to the amount of protein in your sample. Make sure to include a lane with molecular weight standards (PageRuler Prestained Protein Ladder™ (Fa. Fermentas)) to determinate the molecular weight of your sample
- Connect the power lead and run the stacking gel with 10 mA until the blue dye front enters the separating gel
- Raise amperage up to 20 mA for running the separating gel
- When the distance of the lowest molecular weight standard lane to the gel end is down to 0.5 cm stop the electrophoresis by turning off the power supply
NADH-Assay
- This method has been used for measurement of intracellular NADH concentration.
- Protocol:
- Inoculate an overnight culture (30 mL with 1 mL of pre-culture)
- Centrifugate (5 min at 5000 g) of 6-10 mL overnight culture (OD 4-6), adjust volume between different samples for the approximation of the number of cells
- Discard supernatant and wash pellet 3 times with 1 mL of PBS buffer
- Resuspend pellet in 1 mL PBS buffer
- Cell disruption by Ribolysation (3 x 30 sec at 6500 rpm)
- Centrifugation for 10 min at maximum speed
- Store supernatant at - 20 ° C or direct measurement with Tecan Infinite® M200 platereader
- Tecan Infinite® M200 platereader parameters:
- Sample volume = 100 μL clear supernatant
- Excitation = 340 nm
- Emission = 460 nm
- Concentration calculation by NADH calibration curve
Hexadecan Assay
- This assay has been used to measure cell membrane hydrophobicity.
- Protocol
- Inoculate an overnight culture (30 mL with 1 mL of pre-culture)
- Centrifugate (5 min at 4000 g) of 2 mL overnight culture (OD 4-6)
- Discard supernatant and wash pellet 3 times with 1 mL of PBS buffer
- Resuspend pellet in 1 mL 0,9% NaCl and measurement of OD600
- Add x μL of washed cells to 0,9% NaCl for a final volume of 3 mL . Final OD600 should be approximately 0,3 (denoted as A0, calculate exact value)
- Add 3 mL Hexadecan and vortex for 60 sec
- Incubation for 15 min
- Discard the upper organic phase and measure OD600 of the aqueous phase (denoted as A)
- Hydrophobicity can be calculated using the equation: affinity [%] = 100 x [1 – (A/A0)]
Cold osmotic shock
- Release of periplasmic protein fraction from E. coli by cold osmotic shock
- Modified protocol from Neu & Heppel (1965)
- Centrifuge E. coli cell suspension for 5 min at 14,000 g (4 °C) to collect the cells
- Discard the entire supernatant
- Resuspend the cells in ice-cold cell fractionating buffer 1. The resulting volume should be 1/4 of the former suspension volume
- Incubate for 20 min on ice. Invert the suspension at regular intervals to counteract sedimentation
- Centrifuge the cell suspension for 15 min at 14,000 g (4 °C)
- Discard the entire supernatant
- Resuspend the cells in ice-cold cell fractionating buffer 2. The resulting volume should be 1/4 of the former suspension volume
- Incubate for 10 up to 20 min on ice under regular invertion
- Centrifuge the cell suspension for 15 min at 14,000 g (4 °C)
- Save the supernatant, which contains the periplasmatic proteins and membrane proteins when using Cell fractionating buffer 2.2 and 2.3
- If the periplasmatic protein fraction is turbid, re-centrifuge and filter it through a 0.2 µm filter
Cultivation
Cultivation in liquid LB-Medium
Media
Chloramphenicol stock solution
- Solubilize 20 mg mL-1 Chloramphenicol in 100 % Ethanol
- Store at -20 °C
DNA loading buffer
- 50 % (v/v) glycerol
- 1 mM EDTA
- 0.1 % (w/v) bromphenol blue
- Solve in ddH2O
LB medium
- 10 g Trypton
- 5 g yeast extract
- 10 g NaCl
- 12 g Agar-Agar (for plates)
- Adjust pH to 7.4
M9 minimal medium
- For 1 L M9 mineral medium 867 mL sterile water is needed(for plates add 16 g Agar-Agar as well)
- Then add in the following order:
- 00 µL 1 M CaCl2
- 1 M CaCl2
- 1 M CaCl2-H20
- 14.70 g/100 mL
- 100ml 5x M9 salt solution
- 10x M9 salt solution
- Na2HPO4-2H2O 75.2 g/L
- KH2PO4 30 g/L
- NaCl 5 g/L
- NH4Cl 5 g/L
- 10x M9 salt solution
- 20 % glucose
- 20 % (w/v)glucose 200 g/L
- For 500 mL stock solution add 100 g glucose to 440 mL water. Autoclave for 15 at 121°
- 1 M MgSO4 </p>
- 1 M MgSO4-7H20 24.65 g/100 mL
- 1 mL Biotin (1 mg/mL)
- Biotin (1 mg/mL) 50 mg/50 mL
- For 50 mL stock solution dissolve 50 mg biotin in 45 mL water. Add water to a final volume of 50 mL. Sterilize the solution over a 0.22-µm filter. Prepare 1 mL aliquots and store at -20°.
- 1 mL Thiamin (1 mg/mL)
- Thiamin-HCl (1 mg/mL) 50 mg/50 mL
- For 50 mL stock solution dissolve 50 mg thiamin-HCl in 45 mL water. Add water to a final volume of 50 mL. Sterilize the solution over a 0.22-µm filter. Prepare 1 mL aliquots and store at -20°.
- 10 mL 100x trace elements solution
- 100x trace elements solution
- EDTA 5 g/L 13.4 mM
- FeCl3-6H2O 0.83 g/L 3.1 mM
- ZnCl2 84 mg/L 0.62 mM
- CuCl2-2H2013 mg/L 76 µM
- CoCl2-H2O 10 mg/L 42 µM
- H3BO3 10 mg/L 162 µM
- MnCl2-4H20 1.6 mg/L 8.1 µM
- Dissolve 5g EDTA in 800 mL water and adjust the pH to 7.5 with NaOH. Then add the other components in the quantities mentioned below and add water to a final volume of 1L. Sterilize the solution over a 0.22µm filter</p>
- 100x trace elements solution
- Unknown
- 498 mg FeCl3(anhydrous)
- 84 mg ZnCl2
- 765 µL 0.1 M CuCl2-2H20 1.70 g/100 mL
- 210 µL 0.2 M CoCl2-H2O 4.76 g/100 mL
- 1.6 mL 0.1 M H3BO3 0.62 g/100 mL
- 8.1 µL 1M MnCl2-4H20 19.8 g/100 mL
SOC medium
- Add the following components for 900 ml of distilled H2O:
- 20 g Trypton
- 5 g Bacto Yeast Extract
- 2 mL of 5 M NaCl
- 2.5 ml of 1 M KCl
- 10 ml of 1 M MgCl2
- 10 ml of 1 M MgSO4
- 20 ml of 1 M glucose
- Adjust to 1L with distilled H2O. Sterilize by autoclaving.
Buffers & Solutions
Biotin 500x Stock
- Dissolve 20 mg biotin in 100 mL of 0.05 M NaOH solution and filter sterilize
- Store at 4 °C
- Durable for one year
TAE-Buffer
- 1 L of 50x TAE buffer
- 242.48 g Tris
- 41.02 g Sodiumacetate
- 18.612 g EDTA
- Adjust pH to 7.8 with acetic acid
- Solve in dH2O
- Dilute 20 mL 50x stock in 1L dH2O for 1x Buffer for PAGE
Cell Fractioning Buffers
CFB 1 (pH 8)
- 0.2 M Tris
- 200 g L -1 sucrose
- 0.1 M EDTA
CFB 2.1 (pH 8)
- 0.01 M Tris
- 0.005 M MgSO4
CFB 2.2 (pH 8)
- 0.01 M Tris
- 0.005 M MgSO4
- 1% Trition
- 2% SDS
CFB 2.3 (pH 8)
- 0.01 M Tris
- 0.005 M MgSO4
- 1% Trition
- 0.2% SDS
CFB 2.4 (pH 8)
- 0.01 M Tris
- 0.005 M MgSO4
- 1% Trition
- 1% SDS