Team:DTU-Denmark/Notebook/2 July 2013

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02 July 2013

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208 lab


Main purposes today


Create dilution series for biolector experiment.

Who was in the lab


Helen, Natalia, Ariadni

Procedure


Weighed:

  • 2.3187g Ammonium acetate
  • 0.2557g Sodium nitrate
  • 0.2080g Sodium nitrite

Diluted each in 10mL M9 medium.

Created dilution series as in https://docs.google.com/spreadsheet/ccc?key=0AkM9Z7voM1otdFZ5SWstOWpURGs2S3M5bElMMllkenc

Each solution was diluted with media.

Salt mM
Sodium Nitrate 300
Sodium Nitrate 30
Sodium Nitrate 3
Sodium Nitrate 0.3
Sodium Nitrite 300 (Master)
Sodium Nitrite 30
Sodium Nitrite 3
Sodium Nitrite 0.3
Sodium Nitrite 0.03
Ammonium Acetate 3000
Ammonium Acetate 300
Ammonium Acetate 30
Ammonium Acetate 3

208 lab afternoon


Main purposes today


Performing Colony PCR to verify if transformants contain proper construct (signal peptide, GFP, RFP - in plazmid pZA21).

Who was in the lab


Gosia, Ariadni

Procedure


Among colonies which grew after transformation 11 colonies were chosen to be verified by colony PCR. Plates with transformants and colonies checked by colony PCR are signed:

  1. T2 U2 Sec - colonies 01, 02, 03
  2. T2 U2 TAT2 - colonies 01, 02, 03
  3. T2 U3 TAT3 - colonies 01, 02, 03, 1a, 2a

T2 U2 Sec stands for 2nd transformation, 2nd USER reaction, Sec signal peptide TAT2 and TAT3 is TAT signal peptide

All checked colonies were plated on a new plate, overnight incubation at 37°C.

PCR master mix for 12 samples (one additional just in case) was done according to Colony PCR method. Forward primer for colonies with Sec signal (PCR tubes signed: 1, 2, 3) peptide was primer for GFP Sec FW (5a). Forward primer for colonies (PCR tubes signed: 4, 5, 6, 7, 8, 9, 10, 11) with TAT was GFP TAT FW (4a). Reversed primer for all colonies was RFP RW (6b n).

In positive colonies amplified fragment of lenght about 1550 bp is expected.

PCR program (IGEM_CO)

  1. 98°C for 2:00
  2. 98°C for 0:10
  3. 67°C for 0:05
    • ramp 0.1°C/s until 62°C
  4. 72°C for 1:30
  5. Go to number 2 - repeat 50
  6. 72°C for 5:00
  7. 4°C for 10:00:00
  8. End


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