Team:Aachen/Notebook/Protocols

From 2014.igem.org

(Difference between revisions)
(Electroporation)
(Agar Chips)
Line 61: Line 61:
# cut out the chips with a scalpel
# cut out the chips with a scalpel
# put ever two chips into a labeled petri dish
# put ever two chips into a labeled petri dish
 +
# incubate for 1h by 37°C
= Transformation =
= Transformation =

Revision as of 15:27, 2 October 2014

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)

SOC

  1. components
  • 0,5 % yeast extract
  • 2 % tryptone
  • 10 mM NaCl
  • 2,5 mM KCl
  • 20 mM MgSO4
  1. fill up with deionized water
  2. adjust to pH 7,5 with NaOH
  3. after autoklave add 20 mM sterile filtered glucose

Agar Chips

Cell preparation

  1. over night culture of sensor cells (50 ml in a 250 ml flask with) max. 16h
  2. centrifuge all 50 ml by 3000 xg for 10 min.
  3. discard the supernatant
  4. resuspend the pellet in 1 ml warm medium (RT)


Agar preparation

  1. autoclave 50 ml medium with 1,5 % agarose
  2. cool it down to 45 °C in a water bath


Chip production

  1. mix the cooled dowm medium with the cells by inverting gently
  2. pour it in the chip form without bubbles
  3. wait for ca 20 min till it is solid
  4. cut out the chips with a scalpel
  5. put ever two chips into a labeled petri dish
  6. incubate for 1h by 37°C

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

  1. add 1 μl plasmid to electrocompetent cells
  2. put DNA/ cell suspension in electroporation cuvette
  3. wipe dry the electroporator
  4. use small plastic pipette to place the cells
  5. puls: 2,5 kV, 200-400Ω, 25 μF (for E.coli)
  6. immediatly add 1 ml LB and incubate for 2h by 37 °C
  7. plate 50 μl on selectiv medium plate
  8. centrifuge the rest (3000 xg, 20 min), discard supernatant, resuspend the pellet in 50 μl and plate it on selectiv medium plate

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