Basic Protocols

because I forget everything

Sterilization (aka autoclaving)

We’re using pressure cookers for sterilization.

Put some petri dishes on the bottom for support, fill up with distilled water so that the petrish dishes are not yet covered. (The idea here is for the water not to reach the stuff you want to sterilize, and them not touching the bottom of the pan either).

Cover the open parts of the stuff to be sterilized with aluminium foil (so that you don’t contaminate them later), and put them into the pressure cooker.

Put on the electric stove, max power, wait until the pressure valve starts to release the pressure. Reduce power, and wait 20 minutes. Turn stove off, release pressure via valve, open.

Ampicillin stock solution

Add 5ml 96% ethanol to 500mg of amicillin in a sterile vial and stir. keep in deep freezer, -20°C. After doing so, disperse into 1ml or 0.5ml aliquots for storage.

This will result in an ampicillin stock of 100mg/ml. The final concentrations we’re aiming for is 100µg/ml. To achieve this, add 1/1000th of the overall quantity of the stock ampicillin solution, say for 100ml add 100µl.

Arabinose stock solution

Take L(+) Arabinose in powerder form, add 5ml of distilled water / g, wait until the arabinose dissolves. Then push the solution through a 0.2µm filter into a sterile vial. store in the regular freezer, +8°C.

This will result in a 20% m/m arabinose stock soltuon, that is 200mg / ml. The final concentration we’ll be looking for is 5mg / ml, say for 100ml overall soltuon 2.5ml of stock arabinose solution is needed.

LB (Liquid Broth)

for 1l of LB, 1 liter of distilled water to:

  • Tryptone: 10g
  • Yeast Extract: 5g
  • NaCl: 10g
  • for LB agar, add 15g agar
  • for top agar: add 7.5g agar

then autoclave

the same for 100ml of LB:

  • Tryptone: 1g
  • Yeast Extract: 0.5g = 500mg
  • NaCl: 1g
  • for LB agar, add 1.5g agar
  • for top agar: add  0.75g = 750mg agar

then autoclave

LB / ampicillin

for LB with ampicillin, add 1/1000th of the overall quantity ampicillin solution. prepare as above, after it cools below 50°C:

  • for 1l LB = 1000ml LB, add 1ml of ampicillin stock solution
  • for 100ml LB, add 100µl of ampicillin stock solution

this will result in a 100µg/ml final ampicillin concentration

LB / ampicillin / arabinose

for LB with ampicillin and arabinose, first add ampicillin as described above. then add:

  • for 1l LB = 1000ml LB, add 25ml arabinose stock solution
  • for 100ml LB, add 2.5ml arabinose stock solution

this will result in a 5mg/ml final arabinose concentration

One Response to “Basic Protocols”

  1. bio.display » first successful DNA transformation in the new lab Says:

    […] also summed up most of the basic protocols, ways to grow bacteria and the DNA transformation steps as well – because I always forget […]

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