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From 2014.igem.org

Lab Protocols

Protocol 1 - Produce LB broth

Materials and Equipment

LB broth mix, sterile water, weighing scales, weighing boat, conical flask, measuring cylinder.

Time

Prep: 5 minutes
Run: 2 hours

Procedure

Weigh out 2g of LB broth mix in a weighing boat and add into a 250ml conical flask.
Measure 100ml sterile water into a measuring cylinder and dispense into the same conical flask.
Swirl.
Stopper the flask with cotton wool and foil.
Label on autoclave tape. Format 'iGEM, Room Number'.
Move the flask(s) to the autoclave to be sterilised.

Protocol 2 - Produce LB Agar

Materials and Equipment

LB Agar mix, sterile water, weighing scales, weighing boat, conical flask, measuring cylinder.

Time

Prep: 5 minutes
Run: 2 hours

Procedure

Weigh out 3.5g of LB agar mix in a weighing boat and add into a conical flask
Measure 100ml sterile water into a measuring cylinder and dispense into the same conical flask.
Swirl.
Stopper the flask with cotton wool and foil.
Label on autoclave tape. Format 'iGEM, Room Number'.
Move the flask(s) to the autoclave to be sterilised.

Protocol 3 - Make overnight starter cultures

Materials and Equipment

Stripette, bunsen burner, sterile loop, falcon tubes, LB broth.

Time

Prep: 10 minutes
Run: 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 4 - 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 minutes
Run:5 hours

Procedure

1. 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

2. 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.

3. 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?

4. 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 5 - Mini-prep

Materials and Equipment

Ice, starter cultures, mini-prep kit, centrifuge, P100 pipette, weighing scale, Virkon

Time

Prep:
Run:

Protocol

1. 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

2. 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

3. 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

4. Neutralise cells

Add 350μl of N3 neutralisation buffer
Once the reaction is complete, the liquid will turn clear/white

5. 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 freezer

Protocol 6 - 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 minutes
Run: 1 hour

Protocol

C60 - This is an Ethidium bromide lab. Always wear gloves.

1. 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.

2. 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.

3a .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.

3b. 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.

4. 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 7 - Pouring Plates

Materials and Equipment

Sterile hood, Flask, Agar mix, dH₂O, antibiotic, empty plates, P200, tips.

Time

Prep:15 minutes
Run:20

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 8 - Measure concentration of DNA with NanoDrop2000

Materials and Equipment

Nanodrop, P10, Tips, dH₂O, Paper towel.

Time

Prep: 5 minutes
Run: 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 9 - 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, Ethanol

Time

Prep:5 minutes
Run: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 10 - Blunt End Ligation

Materials and Equipment

Eppendorfs, P10, tips, Reaction buffer, dH₂O, Restricted DNA, T4 Ligase.

Time

Prep: 10 minutes
Run: 10 minutes

Protocol

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 11 - Chemical Transformation

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 minutes
Run: 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 12 - sticky end ligation

Materials and Equipment

Eppendorf, p10, tips, reaction buffer, dH₂O, restricted DNA, T4 Ligase.

Time

Prep:5 minutes
Run:30minutes

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 13 - Restriction Digest

Materials and Equipment

Eppendorf, p10, p100, tips, restriction enzymes, ice, heat block.

Time

Prep: 5 minutes
Run: 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 14 - Glycerol stocks

(Allows stocks of cells to be kept in the -80)

Materials and Equipment

P1000 pipette, tips, eppendorf tubes, microwave, 100% glycerol, overnight cultures of cells

Time

Prep: 5 minutes
Run: 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.


μ (mu) °
Make LB broth protocol