(chemically competent cells,
protocol from Allison Lab)
1.
Streak a LB plate with DH5a stock cells (or strain of choice) & incubate overnight at 37oC
2. Inoculate
FOUR Corex tubes containing 5ml LB with the repeated picks of same DH5a colony. Incubate overnight
in a shaker at 37oC.
3. Warm FOUR 1L
flasks and 1L of LB overnight in the 37oC incubator
4. Aliquot the
warmed LB into the warmed 1L flasks (transfer ~10ml LB medium for use as
blank). Inoculate each flask with a
fresh 5mL culture of DH5a. Incubate with shaking at 37oC
until A650 reaches 0.15 to 0.2 (CLR Lab Spec – NO path check). This step should take approximately 1 to 3
hours. When desired cell OD obtained,
chill cultures on ice and turn on the centrifuge to cool.
IMPORTANT
- Perform all of the following steps maintaining the cells ON ICE!
5. Pool the
cultures and mix well. Divide the
cultures among SIX 85 ml centrifuge bottles.
Spin for 8 minutes at 6000rpm at 4oC (you will need to do two
separate spins in each bottle to collect all cells). Discard supernatant and gently resuspend each pellet in 14ml of
cold 0.1M CaCl2 by swirling and washing pellets with a pipet. Resuspension may take 10 minutes. Incubate cells 20 minutes on ice, distribute
to FOUR 85 ml centrifuge bottles and pellet as before.
6. Gently
resuspend each pellet in 10ml of cold 0.1M CaCl2 at this point the
cells can be held on ice overnight.
7. Add 9ml cold
80% glycerol to the total volume of cells and mix well. Distribute 600ul aliquots to sterile
Eppendorf tubes. Flash freeze in liquid
nitrogen and store at –80oC.
8. Calculate
Cell Transformation Efficiency: Perform
E.coli transformation protocol with a known mass of plasmid DNA. Competency is calculated as number of colony
forming units (CFUs) from 1ml of cells per mg plasmid DNA.