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Team:Stockholm/16 September 2010
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[[image:ColPCR_Tra10*SH_16sep.png|200px|thumb|right|'''Colony PCR gel verification of N-Tra10⋅SOD⋅His clones.'''<br />4 μl λ; 5 μl sample.<br />λ = O'GeneRuler 1 kb DNA ladder.]] | [[image:ColPCR_Tra10*SH_16sep.png|200px|thumb|right|'''Colony PCR gel verification of N-Tra10⋅SOD⋅His clones.'''<br />4 μl λ; 5 μl sample.<br />λ = O'GeneRuler 1 kb DNA ladder.]] | ||
[[image:ColPCR_pEX.SOD_16sep.png|200px|thumb|right|'''Colony PCR gel verification of pEX.SOD clones.'''<br />4 μl λ; 5 μl sample.<br />λ = O'GeneRuler 1 kb DNA ladder.]] | [[image:ColPCR_pEX.SOD_16sep.png|200px|thumb|right|'''Colony PCR gel verification of pEX.SOD clones.'''<br />4 μl λ; 5 μl sample.<br />λ = O'GeneRuler 1 kb DNA ladder.]] | ||
| + | |||
| + | '''Gel 1'''<br /> | ||
| + | 1 % agarose, 110 V | ||
| + | |||
| + | Expected bands | ||
| + | *pSB1K3.N-TAT⋅SOD⋅His (TAT): 848 bp | ||
| + | *pSB1C3.SOD⋅His (pC.SH): 815 bp | ||
| + | |||
| + | '''Gel 2'''<br /> | ||
| + | 1 % agarose, 110 V | ||
| + | |||
| + | Expected bands | ||
| + | *pSB1K3.N-Tra10⋅SOD⋅His (Tra10): 878 bp | ||
| + | *pSB1C3.SOD⋅His (pC.SH): 815 bp | ||
| + | |||
| + | '''Gel 3'''<br /> | ||
| + | 1 % agarose, 110 V | ||
| + | |||
| + | Expected bands | ||
| + | *pEX.SOD: 678 bp | ||
| + | *pEX.RFP: 862 bp, 1010 bp, 1124 bp, | ||
Revision as of 23:14, 20 September 2010
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Contents |
Andreas
Assembly of new parts
Gel verification of part digestions
Ran gels of digestion samples in parallel with undigested samples to verify successful digestions and insert sizes.
Gel 1
&4 μl λ; 3 μl sample.
λ = O'GeneRuler 1 kb DNA ladder.
1 % agarose, 110 V
Gel 2
&4 μl λ; 3 μl sample.
λ = O'GeneRuler 1 kb DNA ladder.
1 % agarose, 110 V
Results
Successful digestion with corresponding bands for all digested samples. N-TAT and N-Tra10 not verified, since gel was run too far, but plasmid linearization should indicate successful digestion.
Most samples show somewhat incomplete digestion. For digestions with FastDigest enzymes, this may be an indication of old/inactive enzymes. Especially PstI should be analyzed for activity.
Colony PCR
Picked new colonies for colony PCR from 14/9 plates:
- pSB1K3.N-TAT⋅SOD⋅His: TAT⋅SH 1-5
- pSB1K3.N-Tra10⋅SOD⋅His: Tra10⋅SH 1-5
- pEX.SOD 1-4
Standard colony PCR settings.
- Elongation: 1:30
Gel verification
4 μl λ; 5 μl sample.
1 kb λ = O'GeneRuler 1 kb DNA ladder; 50 bp λ = GeneRuler 50 bp DNA ladder.
4 μl λ; 5 μl sample.
λ = O'GeneRuler 1 kb DNA ladder.
4 μl λ; 5 μl sample.
λ = O'GeneRuler 1 kb DNA ladder.
Gel 1
1 % agarose, 110 V
Expected bands
- pSB1K3.N-TAT⋅SOD⋅His (TAT): 848 bp
- pSB1C3.SOD⋅His (pC.SH): 815 bp
Gel 2
1 % agarose, 110 V
Expected bands
- pSB1K3.N-Tra10⋅SOD⋅His (Tra10): 878 bp
- pSB1C3.SOD⋅His (pC.SH): 815 bp
Gel 3
1 % agarose, 110 V
Expected bands
- pEX.SOD: 678 bp
- pEX.RFP: 862 bp, 1010 bp, 1124 bp,
