Team:Stockholm/8 August 2010

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Contents

Andreas

Cloning

Colony PCR

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. 1 % agarose, 110 V, 35 min
  2. 1 % agarose, 90 V, 30 min

Results

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.
  1. Bands seem identical in size. Highly probable that my yCCS clones are incorrect.
  2. 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.
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