User:AndreasConstantinou/28 June 2010

From 2010.igem.org

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

1. Colony carrying pEX vector were picked from agar plate and resuspended in 10 ul LB.
   • Left to incubate in room temperature.
2. 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
3. Amplification by PCR
   • Hot-start: 95°C - 1 min
   • 30 cycles
   1. Denaturation: 95°C - 30 s
   2. Gradient (annealing): 50°C, 55°C, 60°C - 30 s
   3. 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