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 10' @95°C 30 × (30" @95°C; 15" @64; text@72°C); optional: 10' @72°C ∞ 4°C extension time text = (kb insert length) × 1' (1 min)

1. prepare 96-well "Lysis" plate:
   1. multi-dispense 50 µl H2O into each well
   2. pick 8 colonies of one assembly into the wells of one column (like A1-H1 or A2-H2)
   3. copy 4 µl per well from this column onto a backup LB plate with Ampicilin (using a 8-channel pipette)
      • label the plate appropriately beforehand (like "A1 -> H1")
   4. proceed to next column for next assembly
2. prepare PCR plate:
   1. multidispense 10 µl PCR mastermix into each well
   2. copy 1 µl per well from lysis plate to PCR plate (using multi-channel pipette)
   3. seal PCR plate with adhesive tape
3. run PCR program
4. put backup plates @ 37 °C

II Agarose Gel

1. prepare a large 0.7 - 1.5 % Agarose gel (depending on fragment size)
2. multi-dispense 2 µl loading buffer into each well of the PCR plate
3. load gel with multi-channel pipette
4. run, analyze, enjoy!

Notes

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