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From 2010.igem.org

Contents 1 Andreas 1.1 Colony gradient PCR verification of pEX vector 1.1.1 Procedures 1.1.2 Results 2 Hassan 3 Mimmi 3.1 pMA 3.1.1 Designing primers for verification 3.2 pEX 3.2.1 Start over night culture for miniprep.

Andreas

Colony gradient PCR verification of pEX vector

Gradient PCR was run to optimize the colony PCR settings for designed pEX verification primers (pEXf and pEXr). Products analyzed by agarose gel electrophoresis.

Procedures

PCR settings

• Primers:
  • pEXf forward primer (CGG CTC GTA TAA TGT GTG GAA TTG)
  • pEXr reverse primer (CGT TCA CCG ACA AAC AAC AG)
• Gradient: 50°C, 55°C, 60°C
• Elongation time: 2 min 30 s
  • Expected amplicon size: 116 bp (pEXf) + 103 bp (pEXr) + 1188 bp (insert) = 1407 bp

Agarose gel analysis

• 1% agarose gel
• 2 ul sample
• 170 V, 20 min

Results

Figure to be added later.

Hassan

May the "http://www.pathguide.org" guide us through the darkness :)

Mimmi

pMA

Designing primers for verification

• Get vector sequence from iGEM part registry

• To be able to test in AmplifX, reajust sequence:

• In program: Design primers
  • primer length
  • max Tm difference 2°C
  • F primer: 234-284bp
  • R primer: 634-684bp

• Choose a primerpair with the same Tm

• "Run PCR" -> looking good! No dimers or unspecific binding

pEX

Start over night culture for miniprep.

• Mix one colony in 5ml LB containing Amp. (100µg/ml) in a falcon tube

• Leave ON in 37°C, 250rpm with some what opened lid.