Team:Stockholm/8 August 2010
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==Andreas== | ==Andreas== | ||
+ | |||
+ | ===Cloning=== | ||
+ | |||
+ | ====Colony PCR==== | ||
+ | [[Image:ColPCR_yCCS_8aug.png|200px|thumb|right|Comparison of colony PCR results of pSB1A3.yCCS A & B and pSB1C3.BBa_J18932.<br> | ||
+ | '''Loading:''' 1 kb λ, yCCSA A, yCCSA B, yCCSB A, yCCSB B, BBa_J18932 A, BBa_J18932 B<br> | ||
+ | 3 μl &lambda, 6 μl sample.]] | ||
+ | [[Image:ColPCR_IgGp_8aug.png|200px|thumb|right|Colony PCR gel verification of IgG protease.<br> | ||
+ | '''Loading:''' A, B, 1 kb &lambda, C, D, blank.<br> | ||
+ | 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'''<br> | ||
+ | *22.5 μl dH<sub>2</sub>O | ||
+ | *1 μl VF2 | ||
+ | *1 μl VR | ||
+ | *0.5 μl cell suspension | ||
+ | *illustra Ready-to-Go PCR Beads | ||
+ | |||
+ | ::'' '''Negative control (18):''' Blank'' | ||
+ | |||
+ | '''PCR settings''' | ||
+ | {|border="1" cellpadding="2" cellspacing="0" | ||
+ | |'''1) Denaturation:''' | ||
+ | |colspan="2"|95 °C - 10:00 | ||
+ | |- | ||
+ | |'''2) Denaturation:''' | ||
+ | |95 °C - 0:30 | ||
+ | |rowspan="3"|x30 | ||
+ | |- | ||
+ | |'''3) Annealing:''' | ||
+ | |55 °C - 0:30 | ||
+ | |- | ||
+ | |'''4) Elongation:''' | ||
+ | |72 °C - 1:45 | ||
+ | |- | ||
+ | |'''5) Finish:''' | ||
+ | |colspan="2"|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. | ||
+ | |||
+ | {{Stockholm/Footer}} |
Latest revision as of 10:47, 26 October 2010
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 % 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.