LB and LB/LBAmp plates

9/9/05

For LB:

Weigh 20g LB broth mix (USB 75852), add to 1L ddH2O in a beaker.

Stir and add 1mL 1M NaOH to pH to 7.5.

Check pH with paper, not electrode as protein can clog the electrode

Aliquot by pouring 250mL into a 500mL bottle to autoclave. Screw bottle top on loosely and cover with foil and tape to hold it on.

Autoclave in an autoclave pan with 1” water in the bottom for 20-30’ at 121 degrees.

Allow to cool before tightening caps.

For LB/LB Amp plates:

Unpack the petri dishes (Falcon 1029) you will need and SAVE THE SLEEVE.

Weigh 17.5g LBAgar mix (USB 75851).

Add to 500mL ddH2O in a 2L flask. Be forewarned: the agar does not go into solution so you will not be able to pour this sludge into another vessel.

Add 0.5mL 1M NaOH to pH to 7.5.

Cover the top with foil and tape it on.

Autoclave in an autoclave pan with 1” water in the bottom for 20-30’ at 121 degrees.

Cool in a 65 degree water bath or on the bench until you can touch the bottom of the flask without too much pain (=65 degrees). If cooling on the bench, be careful it doesn’t congeal! If it does, you must start over.

If you want LB plates, pour on petri dishes, 25mL/dish (100mm size). You can pour until the bottom is fully covered or pipette. If you want to get rid of the bubbles, you can flame the top but BE CAREFUL and DON’T SET THE BENCH ON FIRE!! Better to just live with the bubbles unless they are really bad.

If you want LB Amp plates, add 25mg of Ampicillin (USB 11259) to 500mL of LB Agar. This gives a final concentration of 50ug/mL. Note that sometimes people use twice this amount of ampicillin. Swirl the flask to thoroughly mix the ampicillin in, then pour onto plates.

Allow plates to cool face up for a few hours.

Mark LB Amp plates with a pen streak on the side of each dish and make sure to also label the sleeve. There are many different antibiotics that can be added to plates. Make sure you know what it is in the plates you are using.

Place plates back in the sleeve, label and DATE (very important), tape closed, and store upside down (to reduce water accumulation on the agar) in the cold room for up to 2 months.

1M NaOH:

MW is 40g/mol

To make 50mL of 1M NaOH, weigh 2g of NaOH pellets, place in 50mL plastic tube and add ddH2O to 50mL. Mix to dissolve. Date the tube and store by pH meter.