Team:Stockholm/8 August 2010

From 2010.igem.org

Andreas

Cloning

Colony PCR

Comparison of colony PCR results of pSB1A3.yCCS A & B and pSB1C3.BBa_J18932.
Loading: 1 kb λ, yCCSA A, yCCSA B, yCCSB A, yCCSB B, BBa_J18932 A, BBa_J18932 B
3 μl &lambda, 6 μl sample.

Colony PCR gel verification of IgG protease.
Loading: A, B, 1 kb &lambda, C, D, blank.
3 μl λ, 6 μl sample.

Picked four colonies from 3/8 IgG protease plate (A, B, C & D). Also chose two plasmid samples of pSB1C3.BBa_J18932 (A & B) for PCR verification, to be compared with the amplified yCCS samples from 7/8.

PCR tubes

22.5 μl dH₂O
1 μl VF2
1 μl VR
0.5 μl cell suspension
illustra Ready-to-Go PCR Beads

Negative control (18): Blank

PCR settings

1) Denaturation:	95 °C - 10:00
2) Denaturation:	95 °C - 0:30	x30
3) Annealing:	55 °C - 0:30
4) Elongation:	72 °C - 1:45
5) Finish:	25 °C - ∞

Gel verification

1 % agarose, 110 V, 35 min
1 % agarose, 90 V, 30 min

Results

Bands seem identical in size. Highly probable that my yCCS clones are incorrect.
Strange band sizes, not corresponding at all to what would be expected from IgG protease. I will redo IgG protease cloning next week.
- Did I forget to load the ladder, or did I load loading dye? That might explain the big blob.

The Faculty of Science at Stockholm University

Swedish Vitiligo association (Svenska Vitiligoförbundet)