PrbbBB:colony pcr v1

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Materials

• 96-well PCR plate
• AmpliTaq DNA Polymerase 5 U/µl (Roche)
• AmpliTaq Buffer 10 x
• dNTP mix 10mM each nucleotide
• ddH2O
• primers:
  • BBa_G00100 (VF2) CRG-internal registry
  • BBa_G00101 (VR) CRG-internal registry

Procedure

II PCR reaction

100µl single reaction 3.5xMaster 10xMaster H2O 76µl 266 760 5x HF Buffer 20µl 70 200 10mM dNTP 2µl 7 20 FW primer 100 µM 0.5µl 1.75 5 RV primer 100 µM 0.5µl 1.75 5 Phusion 1µl 3.5 10 template DNA 0.1µl -- --	PCR Program 30"@98°C 5x (10"@98°C; 15"@Ta; text@72°C); 25x (10"@98°C; text@72°C); 10'@72°C ∞ 4°C extension time text = (kb insert length) × 25" annealing temperature Ta = (primer annealing) + 3°C

II Agarose Gel

Notes

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