Team:Sheffield/LabProtocols
From 2014.igem.org
Lab Protocols
Strains
Escherichia coli DH5α - NEB
Escherichia coli MC1000 - Donated by Dr. Graham Stafford
Antibiotics
Antibiotic | Concentration(μg/ml) |
---|---|
Ampicillin | 100 |
Chloramphenicol | 25 |
Tetracycline | 20 |
Primers
Primer Name | Nucleotide Sequence |
---|---|
pSB1C3 seq f | caactcgagtgccacctg |
PSB1C3 seq r | gcctttttacggttcctg |
TliA seq f | gcagatctgagcaccagc |
TliA seq r | gtccagagcacgaaagac |
KERUS seq f | ggtgatggtcaggatagc |
KERUS seq r | ctgccacaacaacaacac |
GGTGATGGTCAGGATAGC
CTGCCACAACAACAACAC
pSB1C3 seq f: caactcgagtgccacctg – 18-mer, 3412...3429, 56degC
pSB1C3 seq r: gcctttttacggttcctg, 18-mer, 1731...1748, 52degC
TliA seq primer f: gcagatctgagcaccagc 18-mer, 711...728, 56degC
TliA seq r: gtccagagcacgaaagac – 18-mer, 831...848, 54degC
Media and Stock Solutions
Luria-Bertani (LB) Broth
LB Broth was made using a ready mix powder from Sigma, 20g/L in ultrapure water. This consists of: 10 g/L Tryptone; 5 g/L Yeast Extract; 5 g/L NaCl. Autoclaved at 121 oC for 15 min to sterilise.
Luria-Bertani (LB) Agar
LB agar was made using a ready mix powder from Sigma, 35 g/L ultrapure water. This consists of 15 g/L Agar and as in LB Broth. Autoclaved at 121 oC for 15 min to sterilise.
5 x M9
5 x M9 was made using the following up to 1 L of ultrapure water: 64 g Na2HPO4-4H2O; 15 g KH2PO4; 2.5 g NaCl.
Working stock, 1 X M9 was made by adding into up to 1 L of ultrapure water: 200 ml of 5 x M9; 2 ml 1M MgSO4; 20 ml 20% glucose/0.4% Glycerol; 100 μl 1M CaCl2
50 x TAE
50 x TAE was made up with: 121 g Tris Base (Trizma); 28.55 gl acetic acid; 9.3 g EDTA made up in 500 ml ultrapure water.
Working stock, 1 x TAE was made by diluting 10 ml in 500 ml ultrapure water.
10 x TBS
10 x TBS was made up with: 500 ml 1M Tris.HCl, pH 7.4; 300 mM NaCl made up to 1L with ultrapure water.
Working stock, 1 x TBS by diluting 100 ml 10 x TBS up to 1000 ml with ultrapure water.
1 x PBS
1 x PBS was made up using Phosphate buffered saline tablets from Sigma.
One tablet dissolved in 200 mL of deionized water yields 0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4, at 25 °C.
Upper Tris Buffer
Upper Tris Buffer was made up by adding 6.05 g Tris Base in 100 ml deionised water, pH 6.8. Add 0.4 g SDS.
Lower Tris Buffer
Lower Tris Buffer was made up by adding 36.4 g Tris in 100 ml deionised water, pH8.8. Add 0.8 g SDS.
10 x SDS Running Buffer
10 x SDS running buffer was made up by adding: 30.3 g Tris Base; 144.1 g Glycine; 10 g SDS to 1 L deionised water to pH 8.3.
Working concentration, 1 x SDS Running Buffer is made by adding 100 ml of 10 x SDS Running buffer to 10 ml deionised water.
Solubilising lipids
A 10% stock solution of triton-x100 was made by adding 1ml of Triton to 9ml sterile (autoclaved) water and incubated at 37oC, 150 rpm until solubilised.
Filter sterilise and add 10% Triton to Olive oil/Oleic acid in a 2:1 ration. Incubate at 37oC, 150 rpm to solubilise.
Protocol 1: Make overnight starter cultures
Materials and Equipment
Stripette, bunsen burner, sterile loop, falcon tubes, LB broth.Time
Prep: 10 minutesRun: 16 hours
Procedure
- Use a stripette to take 5ml of LB broth from a 250ml conical flask.
- Dispense into a falcon tube.
- Sterilise a metal loop in a flame.
- Take a culture from an agar plate using the sterile loop and put into one of the falcon tubes.
- Agitate.
- Replicate this using scrapings from a clean agar plate and a fresh tube to use as a positive control.
- Put the tubes into the incubator overnight (37c, 150rpm) to grow up the cultures.
Protocol 2: Generate chemically competent E. Coli
Materials and Equipment
LB broth, starter culture, P1000, P200, pipette tips, incubator, cuvettes, spectrophotometer, Virkon, falcon tubes, ice, weighing scales, centrifuge, CaCl2, 20% glycerol, stripette, eppendorf tubes and -80°C freezer.Time
Prep: 40 minutesRun: 5 hours
Procedure
- Grow cells
- Take 1ml starter culture and add to 100ml LB broth.
- Incubate at 37°C, 150rpm.
- Check every hour by testing the optical density at 600nm (OD) using a spectrophotometer to determine whether enough cells are present in the culture.
- 0.600 OD is ideal, this is the point at which the cells are in the exponential growth phase.
- Take 1ml of culture into a cuvette to measure; dispose of this after use in Virkon.
- Remove cells
- Pour 30ml aliquots from the flasks into falcon tubes; 3 tubes per flask.
- Put tubes on ice for approx 10mins; all equipment used from this point on must be cold e.g. pipette tips.
- Weigh the falcon tubes and pair together similar weight tubes for balance in the centrifuge; tubes paired together must weigh within 0.5g of each other.
- Spin at 4°C, 4000rpm for 5mins.
- After the tubes have all been spun, pour off the supernatant to remove the LB broth, leaving cells in a pellet. Put tubes back on the ice.
- Make cells competent
- Add 1ml of CaCl2 to the cells, use the pipette to pull the liquid and cells up and down to resuspend.
- Once resuspended, add another 14ml of CaCl2; 15ml total volume.
- Put back on ice for approx. 10mins to allow cells to acclimatise at the temperature with the CaCl2.
- Re-weigh and pairs tubes again for balance.
- Spin again at 4°C, 4000rpm for 5mins.
- After tubes have been spun, leave on ice for 5mins. Pour off the supernatant?
- Aliquot
- Add 600μl of 20% glycerol to each falcon tube.
- Label eppendorf tubes.
- Aliquot 200μl from each falcon tube into eppendorf tubes (3 per falcon).
- Freeze at -80°C.
Protocol 3: Mini-Prep
Materials and Equipment
Ice, starter cultures, mini-prep kit, centrifuge, P100 pipette, weighing scale, Virkon.Time
Prep: ?Run: ?
Procedure
- Extract cells
- Match up weights of falcon tubes so they are paired within 0.5g.
- Spin down starter cultures in a centrifuge; 5mins, 4°C, 4000rpm.
- Pour off supernatant into Virkon.
- Resuspend cells
- Add 250μl of P1 resuspension buffer to each falcon tube using P1000.
- Resuspend the cells.
- Use pipette to move suspension into separate, labelled eppendorf tubes.
- Lyse cells
- Add 250μl of P2 buffer to each eppendorf tube.
- This will lyse cells - a blue colour will indicate they have been lysed.
- Do not leave for more than 5mins.
- Neutralise cells
- Add 350μl of N3 neutralisation buffer.
- Once the reaction is complete, the liquid will turn clear/white.
- Purify DNA
- Spin down the cells in a centrifuge; 10 mins, 17000g, 4°C.
- Pour supernatant into mini-prep columns.
- Centrifuge columns for 1 min, 17000g, 4°C.
- Discard flow through.
- Add 500μl of PB buffer to each column.
- Centrifuge columns for 1min, 17000g, 4°C.
- Discard flow through.
- Add 750μl PE buffer to each column.
- Centrifuge columns for 1min, 17000g, 4°C.
- Pour off supernatent.
- Centrifuge columns for 1min, 17000g, 4°C.
- Discard bottom of the mini-prep.
- Move column to a new labelled eppendorf.
- Add 50μl of elution buffer.
- Centrifuge columns for 1min, 17000g, 4°C.
- Discard column.
- Immediately place on ice; store in the B56 sewer sample freeze.
Protocol 4: Run a gel
Materials and Equipment
Agarose mix, TAE buffer, Flask, Microwave, Ethidium bromide, P10, Tips, Autoclave tape, Gel tray, Comb, Buffer tray, Loading buffer, Dna, Eppendorf, Transilluminator.Time
Prep: 30 mintuesRun: 1 hour
Procedure
- Make up a 1% agarose gel
- Weigh out 0.4g agarose.
- Measure 40ml TAE buffer.
- Add both into a 250ml conical flask.
- Swirl.
- Microwave on full for 2 minute, swirling at intervals to ensure all the agarose has dissolved.
- Run side of flask under tap to cool, until comfortable to hold in a gloved hand.
- Prepare the gel
- Add 1μl ethidium bromide to the conical flask containing the melted agarose.
- Pour this solution smoothly into a gel tray (sealed with autoclave tape) and add a comb which allows for either 8 or 13 DNA samples to be run at once.
- Leave the gel to set for approximately 15 - 30 minutes.
- Once the gel is set remove the comb and autoclave tape.
- Place the gel tray inside the buffer tray and fill the remaining space with TAE buffer.
- Load 'checking' samples
- Load 5μl of 1kn ladder into the first well of the gel.
- Pipette 2μl 5x loading buffer onto a sheet of parafilm.
- pipette 8μl DNA onto the loading buffer and pipette up and down to mix.
- Resting the pipette tip on the back of the well, load 8μl of sample.
- Repeat for each sample.
- Run the Gel at 100v for 60 minutes.
- Load 'extraction' samples
- Load 5μl of 1kn ladder into the first well of the gel.
- Add 5x loading buffer into each sample, to give a final ratio of 1:4.
- Resting the pipette tip on the back of the well, load as much sample as possible.
- Repeat for each sample.
- Visualise
- Remove the agarose gel from the gel tray and place in the trans-illuminator.
- Open UVP (on the computer desktop).
- Select the type of gel and the exposure time.
- Press capture to take an image.
- Save image to the iGEM2014 folder.
Protocol 5: Pouring Plates
Materials and Equipment
Sterile hood, Flask, Agar mix, dH2O, antibiotic, empty plates, P200, tips.Time
Prep: 15 minutesRun: 20 minutes
Procedure
- Use the sterile hood.
- Make up 100ml LB Agar (Protocol 2).
- Heat in the microwave as follows:
- Power setting 4 for 3 minutes.
- Wait for 6 minutes.
- Power setting 4 for 2 minutes.
- Wait to cool (until the flask is warm but comfortable to hold).
- Add 100μl 1/1000 stock of appropriate antibiotic to the agar.
- Pour 4 plates and leave inside the hood to cool and dry.
Protocol 6: Measure Concentration of DNA with NanoDrop 2000
Materials and Equipment
Nanodrop, P10, Tips, dH2O, Paper towel.Time
Prep: 5 minutesRun: 5 minutes
Procedure
- Set up the NanoDrop2000, select “Nucleic Acid”, then “Routine Verification”.
- Spray paper towel with distilled water, then clean metal nodules on metal part and lid.
- Add 1μl of buffer solution to nodule.
- Close lid.
- Select 'Blank' on the programme.
- Reopen lid.
- Add 2μl of DNA Sample.
- Close lid.
- Select 'measure' on the programme.
- Take note of the concentration (in ng/ml).
- Clean nodules again with distilled water.
- Repeat for different DNA sample.
Protocol 7: Transforming Cells To Ensure Competency
Materials and Equipment
Eppendorfs, P1000, P200, P10, Tips, Competent cells, Ligation mix, Ice, Heat block, LB broth, Incubator, Agar plates, Flame, Spreader, EthanolTime
Prep: 5 minutesRun: 1 hour
Procedure
- Using concentration measure by the nanodrop, calculate the volume of DNA sample required: C1V1 = C2V2.
- Where C2 required is 10ng and V2 is 100μl.
- Use the calculated volume of DNA to transform (Protocol 11) chemically competent cells before plating out.
- Calculate the transformation efficiency in colonies per ng of DNA.
Protocol 8: Blunt End Ligation
Materials and Equipment
Eppendorfs, P10, tips, Reaction buffer, dH2O, Restricted DNA, T4 Ligase.Time
Prep: 10 minutesRun: 10 minutes
Procedure
- Make up eppendorfs containing ligation mix, as shown:
- 10μl reaction buffer.
- 8μl water.
- 2μl Cut DNA.
- 1μl Cut plasmid.
- 1μl T4 ligase.
- Flick to mix and leave for ten minutes.
Protocol 9: Chemical Tranformation
Materials and Equipment
Eppendorfs, P1000, P200, P10, Tips, Competent cells, Ligation mix, Ice, Heat block, LB broth, Incubator, Agar plates, Flame, Spreader, Ethanol.Time
Prep: 5 minutesRun: 1 hour 45 minutes
Procedure
- Take an appropriate number of eppendorfs, each containing 100μl of competent E. coli cells.
- Into each one, pipette 5μl of each ligation mix.
- Leave a last one to act as a negative control.
- Put the eppendorfs on ice for 30 minutes.
- Heat shock eppendorfs at 42°C for 30 seconds.
- Put on ice for 2 minutes.
- Add 1ml LB broth to each of the eppendorfs and then incubate at 37°C for 60 minutes.
- Remove cells from incubator and spin down at 13000RPM for 60 seconds.
- Pour off the supernatant and resuspend the cells.
- Near a flame, pipette each of the remaining cells onto individual plates (prepared earlier).
- Sterilise a glass spreader using ethanol (flamed) and spread cells evenly until the surface of the agar appears dry.
- Incubate at 37°C overnight.
Protocol 10: Sticky End ligation
Materials and Equipment
Eppendorf, p10, tips, reaction buffer, dH2O, restricted DNA, T4 Ligase.Time
Prep: 5 minutesRun: 30 minutes
Procedure
- Make up eppendorfs containing the ligation mix, as follows:
- 2μ l reaction buffer.
- 10ng insert.
- 10ng plasmid.
- 1μ l T4 ligase.
- Make up to 20μ l with dH2O.
- Flick to mix and leave for 30 minutes.
Protocol 11: Restriction Digest
Materials and Equipment
Eppendorf, p10, p100, tips, restriction enzymes, ice, heat block.Time
Prep: 5 minutesRun: 1 hour
Procedure
- To each eppendorf add:
- 10 units of restriction enzyme 1 (usually 1μl).
- 10 units of restriction enzyme 2 (usually 1μl).
- 1μg DNA.
- 5μl 10x buffer.
- Make up to 20μl with dH2O.
- Flick to mix.
- Incubate at 37°c for 60 minutes.
- To stop the reaction, heat the sample to an appropriate temperature to inactivate the restriction enzyme used.
- Put on ice.
Protocol 12: Glycerol Stocks
Materials and Equipment
P1000 pipette, tips, eppendorf tubes, microwave, 100% glycerol, overnight cultures of cells.Time
Prep: 5 minutesRun: 5 minutes
Procedure
- Take 800μl of overnight cells using a P1000 pipette and add into separate, labelled eppendorf tubes.
- Microwave the 100% glycerol for 5 seconds.
- Take 200μl of the glycerol using a P1000 pipette and add into each eppendorf tube.
- Place on ice immediately.
- Transfer to the -80°C freezer to be stored.
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