Protocols

Creating Competent Cells

Materials
LB Media, 50ml Falcon Tubes, Ice, Chilled centrifuge, Calcium Chloride (CaCl2), Eppendorf tubes (300ul/tube)

Procedure
1. Inoculate a single colony into 5ml Lb in 50ml falcon tube. Grown O/N @ 37oC
2. Use 1ml to inoculate 100ml of LB in 250ml bottle the next morning.
Shake @ 37oC for 1.5-3 hours.

Or

1. Inoculate a single colony into 25ml LB in a 250ml bottle in the morning
2. Shake @ 37oC for 4-6 hours.

Then…

3. Put the cells on ice for 10mins (keep cold from now on).
4. Collect the cells by centrifugation in the big centrifuge for 3 minutes @ 6Krpm.
5. Decant supernatant and gently resuspend on 10ml cold 0.1M CaCl (cells sensitive to mechanical disruption).
6. Incubate on ice x 20 minutes
7. Centrifuge as in 2.
8. Discard supernatant and gently resuspend on 5ml cold 0.1M CaCl/15%Glycerol.
9. Dispense in microtubes (300ųl/tube). Freeze at -80oC.

Transformation of competent cells

Materials
Competent Cells, Plasmid DNA, Antibiotic Plates
Procedure
1. T haw competent cells on ice
2. 50uL cells enough for 1 transformation
3. Add 1ug of DNA to 50uL competent cells

If biobrick from distribution, resuspend DNA well in 10uL ddH20

4. Add 1uL biobrick DNA to 50uL competent cells
5. Add 1uL RFP control to 50uL competent cells for your control transformation
6. Flick by hand or pipette up and down gently
7. Place cells on ice for 30 minutes
8. Place cells in water bath at 42oC for 40 seconds at 42oC
9. Place cells on ice for 2 minutes
10. Add 0.5mL of LB media and place in incubator for a maximum of 2 hours (37oC/250rpm)42oC (200 µl SOC media can be used to improve transformation efficiency)42oC 11. Label two petri dishes with LB agar and the appropriate antibiotics(s) with the part number, plasmid backbone and antibiotic resistance
12. Plate 50 µl and 500 µl of the transformation onto the dishes, and spread.
13. Incubate the plates at 37ºC for 12-14 hours, making sure the agar side of the plate is up.

If incubated for too long the antibiotics start to break down and un-transformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is excreted by the bacteria, and inactivates the antibiotic outside of the bacteria

You can pick a single colony, make a glycerol stock, grow up a cell culture and miniprep.

Count the colonies on the 20 μl control plate and calculate your competent cell efficiency.