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

I PCR reaction

11µl single reaction 60xMaster 115xMaster H2O 7.5µl 450 862 10x Amplitaq buffer 1.1µl 66 126.5 10mM dNTP 0.22µl 13.2 25.3 VF2 primer 10 µM 0.55µl 33 63.3 VR primer 10 µM 0.55µl 33 63/3 AmpliTaq 5U/µl 0.1µl 6 11.5 template DNA 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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