Protocols

Creating Competent Cells

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

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

Or

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

Then…

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

Transformation of competent cells

Materials Competent Cells - 50ul per transformation DNA - 1ug of DNA per 50ul Antibiotic Plates Procedure · Check which resistance plates you need! Thaw competent cells on ice 50uL cells enough for 1 transformation Add 1ug of DNA to 50uL competent cells If biobrick from distribution, resuspend DNA well in 10uL ddH20 Add 1uL biobrick DNA to 50uL competent cells Add 1uL RFP control to 50uL competent cells for your control transformation pSB1A3 w/ BBa_J04450 Flick by hand or pipette up and down gently Ice for 30 min (roughly) 40s incubation at 42oC in water bath 2 min on ice (minimum - 30 min max) Add 0.5mL of LB media and shake for 30min to 2h Just tape down tube to incubator This is the recovery step SOC media is used if doing cloning in order to improve efficiency Label two petri dishes with LB agar and the appropriate antibiotics(s) with the part number, plasmid backbone and antibiotic resistance Plate 50 µl and 500 µl of the transformation onto the dishes, and spread. This helps ensure that you will be able to pick out a single colony. Use sterile spreader For the control, label two petri dishes with LB agar (AMP). Plate 50 µl and 500 µl of the transformation onto the dishes, and spread. 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.