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.

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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