Team:Bielefeld-Germany/Labjournal/Molecular
From 2013.igem.org
(Difference between revisions)
Line 46: | Line 46: | ||
===Generating electrocompetent cells=== | ===Generating electrocompetent cells=== | ||
*Material: | *Material: | ||
- | *550 mL LB-Medium | + | **550 mL LB-Medium |
- | *1 L cooled bidest. H2O | + | **1 L cooled bidest. H2O |
- | *150 mL cooled 10 % glycerine | + | **150 mL cooled 10 % glycerine |
- | *10 pre-cooled 50 mL Falcons | + | **10 pre-cooled 50 mL Falcons |
*Protocol: | *Protocol: | ||
- | *Inoculate 2x3 mL LB with bacterial stock; incubate over night at 37 °C and 200 rpm | + | **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 | + | **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 | + | **Incubate until OD600 0,4-0,6 |
- | *Cool the culture 15-30 minutes on ice | + | **Cool the culture 15-30 minutes on ice |
- | *Onwards all steps at 4°C | + | **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 | + | **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 | + | **Discard supernatant |
- | *Resuspend pellet in 5 mL cooled bidest H2O (and don't get frustrated while doing it, keep shaking gently) | + | **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) | + | **Pool two suspensions each, add bidest H2O up to 50 mL and centrifugate again (see centrifugation above) |
- | *Discard supernatant | + | **Discard supernatant |
- | *Resuspend pellet in 5 mL cooled bidest H2O | + | **Resuspend pellet in 5 mL cooled bidest H2O |
- | *Add bidest H2O up to 50 mL and centrifugate again (see centrifugation above) | + | **Add bidest H2O up to 50 mL and centrifugate again (see centrifugation above) |
- | *Discard supernatant | + | **Discard supernatant |
- | *Resuspend pellet in 5 mL cooled 10 % glycerine | + | **Resuspend pellet in 5 mL cooled 10 % glycerine |
- | *Transfer suspensions in two 50 mL Falcons and centrifugate again (see centrifugation above) | + | **Transfer suspensions in two 50 mL Falcons and centrifugate again (see centrifugation above) |
- | *Discard supernatant | + | **Discard supernatant |
- | *Add volume of 10 % glycerine that is approximately equal to the volume of the pellet and resuspend | + | **Add volume of 10 % glycerine that is approximately equal to the volume of the pellet and resuspend |
- | *Divide cells in 100 μL aliquots and freeze in liquid N2 immediately | + | **Divide cells in 100 μL aliquots and freeze in liquid N2 immediately |
- | *Store at -80 °C | + | **Store at -80 °C |
Line 127: | Line 127: | ||
*Protocol: | *Protocol: | ||
- | *Inoculate an overnight culture (30 mL with 1 mL of pre-culture) | + | **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 | + | **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 | + | **Discard supernatant and wash pellet 3 times with 1 mL of PBS buffer |
- | *Resuspend pellet in 1 mL PBS buffer | + | **Resuspend pellet in 1 mL PBS buffer |
- | *Cell disruption by Ribolysation (3 x 30 sec at 6500 rpm) | + | **Cell disruption by Ribolysation (3 x 30 sec at 6500 rpm) |
- | *Centrifugation for 10 min at maximum speed | + | **Centrifugation for 10 min at maximum speed |
- | *Store supernatant at - 20 ° C or direct measurement with Tecan Infinite® M200 platereader | + | **Store supernatant at - 20 ° C or direct measurement with Tecan Infinite® M200 platereader |
Revision as of 22:26, 27 September 2013
Protocols
Genetic Engineering
Whole Genome Isolation
- For the isolation following kit has been used: Fermentas GeneJET™ Plasmid Miniprep Kit
- 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 100 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