PrbbBB:colony pcr v1

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Overview

A colony PCR protocol for screening up to 8 clones each for up to 12 different assembly reactions in parallel. Colonies are picked into a 96-well plate with water. The PCR reactions are set up in another 96 well plate and the template DNA is copied over with a multi-channel pipette. During the colony picking, a copy of each clone is put on LB plates. This step can also be performed with a multi-channel pipette (the bugs won't survive for too long in water so it's better to do this whenever you finish a new row).

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

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

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