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Team:Stockholm/8 August 2010
From 2010.igem.org
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#Strange band sizes, not corresponding at all to what would be expected from IgG protease. I will redo IgG protease cloning next week. | #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. | #*Did I forget to load the ladder, or did I load loading dye? That might explain the big blob. | ||
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Latest revision as of 10:47, 26 October 2010
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Contents |
Andreas
Cloning
Colony PCR
Loading: 1 kb λ, yCCSA A, yCCSA B, yCCSB A, yCCSB B, BBa_J18932 A, BBa_J18932 B
3 μl &lambda, 6 μl sample.
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.
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