Highly competent bugs

1. Inoculate 20 ml of TYM with frozen stock in 250ml flask or streak onto LB agar plates

2. Grow to mid-log phase (OD600 is 0.2 to 0.8) app. 4h or pick up colony and grow app. 10h

3. Pour into a 2-liter flask and add 100ml TYM. Flame the tops of both flask

4. Grow to OD 600 is 0.5-0.9 (app. 45min)

5. Add TYM to 500ml (flame both tops) and grow to OD600 of 0.6

6. Put the flask in ice water and shake gently to cool

7. Spin at 2.5 K for 10min at 4⁰ C (use sterile centrifuge bottles)

8. Pour off supernatant and resuspend in 100ml of cold TfB-I by gently shaking in ice

9. Spin at 2.5K for 5min at 4^o

10. Pour off supernatant and resuspend in 20ml of cold TfB-II on ice gently

11. Aliquot the above into prechilled eppendorf tubes in dry ice (100 to 500ml pre tube)

12. Store at -80⁰ C

TYM (store at 4⁰C)

	For 1 liter
-2% Bacto-Tryptone	20g
-0.5% yeast extract	5g
-10mM MgSO4	2.5g

TfB-I (store at 4⁰C)

	For 500ml
-30mM KOAc	5ml of 3M stock
-50mM MnCl2	25ml of 1M stock
-1mM KCl	25ml of 2M stock
-10mM Ca Cl2	5ml of 1M stock
-15% glycerol (v/v)	15ml of 100%

TfB-II (store at 4⁰C)

-10mM NaMOPS (pH 7.0)

-75mM CaCl2

-10mM KCl

-15%glycerol (v/v)

Notes: Use very careful sterile techniques!

Transformation (BRL protocol)

1. Remove competent bugs from -80⁰C and thaw on ice

2. Place the plasmid (1 to 5ng) or ligation mix (1/3 to 1/2 of 20ng mix) in eppendorf tubes and chill on ice. Keep the volume of DNA below 5ml

3. Aliquot 50ml of bugs into the prechilled tubes. Mix gently with the tip of the pipette.

4. Incubate on ice for 30min or longer

5. Heat shock cells for 90sec EXACTLY at 42⁰C

6. Incubate on ice for 2 min

7. Add 450ml of LB

8. Shake at 200rpm at 37⁰C for 30min to 1h to let the drug resistance gene get expressed

9. Plate onto plates containing the selective drug

10. Grow 16-20 (usually overnight)