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Team:Stockholm/28 June 2010
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Revision as of 18:57, 1 July 2010
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Contents |
Andreas
Colony PCR verification of pEX vector
Colony gradient PCR was run to optimize the colony PCR settings for designed pEX verification primers. Products analyzed by agarose gel electrophoresis.
Colony PCR amplification
Materials
- pEXf forward primer (CGG CTC GTA TAA TGT GTG GAA TTG)
- Flanking insert with 116 bp
- pEXr reverse primer (CGT TCA CCG ACA AAC AAC AG)
- Flanking insert with 103 bp
- PuReTaq Ready-To-Go PCR kit (GE Healthcare Life Sciences)
Procedures
- Colony carrying pEX vector were picked from agar plate and resuspended in 10 ul LB.
- Left to incubate in room temperature.
- PuReTaq Ready-To-Go PCR tubes prepared
- 1.5 ul 10uM forward primer
- 1.5 ul 10uM reverse primer
- 1.0 ul template DNA (cell suspension)
- 21 ul dH2O
- Total volume: 25 ul
- Amplification by PCR
- Hot-start: 95°C - 1 min
- 30 cycles
- Denaturation: 95°C - 30 s
- Gradient (annealing): 50°C, 55°C, 60°C - 30 s
- Elongation: 72°C - 2 min 30 s
- Elongation: 72°C - 10 min
Agarose gel electrophoresis analysis
Materials
- 1% agarose gel
- 2 ul sample
- 1.5 ul GeneRuler 1 kb ladder (Fermentas)
Procedures
- 170 V, 20 min
Results
Figure to be added later.
Expected amplicon size: 116 bp (pEXf) + 103 bp (pEXr) + 1188 bp (insert) = 1407 bp
