Making competent cells

Protocol to make competent cells

1.  Make overnight colony of single culture (3mL of TB + Inoculate w/ DH5α)

2.  Make 1:100 dilution and grow to OD550 = 0.5 at 37° in TB (2 hrs with DH1 or 2.5 hrs with HB101)

3.  Transfer to 5-100 ML pre-warmed TB and grow to OD550=0.5 (usually takes 2 hrs)

4.  Chill cells on ice and spin in 4 X 30 mL in pre-chilled centrifuge (4 degrees Celsius) 4000 rpm for 5 minutes

5.  Re-suspend cells (always keep cold) with brief vortexing in ice cold TFB1. 30 microL/100 mL starting culture

6.  Spin immediately with DH1 as in step 5. For HB101, leave on ice for 5 minutes or longer before spinning

*Be quick as possible for thenext few steps, do not let anything thaw out*

7.   Re-suspend pellet gently in 4mL/100 mL starting material in TFB2 (Ice cold)

8.  Aliquot 0.2 ml into 5 ml tubes, put on liquid Nitrogen

9.  Put it in -80 freezer

10. Give yourself a pat on the back

FLOW CHART VERSION