Team:Aachen/Notebook/Protocols

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Contents


Media

LB medium

  1. weight components
  • 5 g/L NaCl
  • 10 g/L tryptone
  • 5 g/L yeast extract
  • (15 g/L agar for plates)
  1. fill up to 1 L with deionized water
  2. mix well by shaking
  3. autoclave
    1. autoclaving tape, caps slightly unscrewed
    2. base of the pot has to be covered with deionized water
    3. close lid
    4. heat level 3 until the pressure valve opens
    5. reduce heat level to 1.5
    6. set timer to 20 minutes
    7. turn heater off
    8. wait until the pressure valve retracts (30-45 minutes)
    9. open, close caps & shake
  4. for plates, wait until you can touch the bottle (<60 °C, clean bench!)
  5. add antibiotics and shake (gloves!)

Hartmans minimal medium (HM)

Agar Chips

Transformation

Heat Shock

  1. thaw cells on ice
  2. add 1 µl of plasmid DNA
  3. incubate on ice for 30'
  4. heat shock at 42 °C for 60
  5. incubate on ice for 5'
  6. add 200 µl of SOC media
  7. incubate at 37 °C for 2 h
  8. plate 20  and 200 µl on plates with the appropiate antibiotic

Electroporation

PCR

Colony PCR

QuikChange

Clonings

Restriction Digest

Ligation

Gibson Assembly

SDS-PAGE

For some SDS-PAGEs we used BioRad ready made gels.

The recipe of the self-made SDS is as follows:

3.5x Buffer

Gels

1mm 12 % RUNNING Gel 1mm 8 % RUNNING Gel 1mm 4 % STACKING Gel
1x 2x 4x 1x 2x 4x 1x 2x 4x
H2O 1.669 ml 3.337 ml 6.674 ml 2.45 ml 4.9 ml 9.8 ml 1.421 ml 2.842 ml 5.684 ml
3.5x Gel Buffer 1.613 ml 3.225 ml 6.45 ml 1.613 ml 3.225 ml 6.45 ml 0.7 ml 1.4 ml 2.8 ml
30 % Acrylamide (37.5:1) 2.344 ml 4.687 ml 9.374 ml 1.563 ml 3.125 ml 6.25 ml 0.329 ml 0.658 ml 1.316 ml
10 % APS 22.5 µl 45 µl 90 µl 22.5 µl 45 µl 90 µl 10.5 µl 21 µl 42 µl
TEMED 2.25 µl 4.5 µl 9 µl 2.25 µl 4.5 µl 9 µl 4.9 µl 9.8 µl 19.6 µl



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