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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:
Run:

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 1min, 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


Make LB broth protocol