PrbbBB:colony pcr v1

Back to all protocols

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 into a deepwell plate with liquid medium which is later used to inoculate over-night cultures for miniprep and cell stock.

Materials

 * two sterile 96-well PCR plates
 * two sterile 96-deepwell plates (preferably 2 ml volume/square well)
 * adhesive tape for plate sealing
 * gas-transmissible adhesive tape for deepwell plate sealing
 * 12-channel 1-10µl pipette
 * multi-dispensing pipette (2 µl minimum) + sterile tips
 * AmpliTaq DNA Polymerase 5 U/µl (Roche)
 * AmpliTaq Buffer 10 x
 * dNTP mix 10mM each nucleotide
 * sterile ddH2O
 * primers:
 * BBa_G00100 (VF2) CRG-internal registry
 * BBa_G00101 (VR) CRG-internal registry
 * liquid LB or 2xTY medium with appropriate antibiotic (Chloramphenicol, Kanamycin, or Tetracycline depending on target plasmid)

Procedure
I PCR reaction


 * extension time text = (kb insert length) × 1' (1 min)


 * 1) prepare 96-well "Lysis" (PCR) plate:
 * 2) sterilize plate and pipette tips
 * 3) multi-dispense 50 µl H2O into each well
 * 4) prepare 96-deepwell "Copy" plate:
 * 5) sterilize plate and pipette tips
 * 6) multi-dispense 100-200 µl sterile LB + appropriate antibiotic into each well
 * 7) colony picking:
 * 8) pick colonies of one assembly with sterile 10 µl tips into...
 * 9) ... the wells of one column (like A1-H1 or A2-H2) on the Lysis plate
 * 10) pipette up and down
 * 11) eject each tip into the same position on the "Copy" deepwell plate
 * 12) proceed to next column for next assembly
 * 13) seal the "Copy" deepwell plate, shake vigorously for 1 min and let it stand at room temperature
 * 14) prepare PCR plate:
 * 15) multi-dispense 10 µl PCR mastermix into each well
 * 16) copy 1 µl per well from lysis plate to PCR plate (using multi-channel pipette)
 * 17) seal PCR plate with adhesive tape
 * 18) run PCR program

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!

III Rescue positive clones
 * 1) inoculate positive clones from "Copy" plate for miniprep (and cell-stock) into a fresh sterile deepwell plate (preferably using a rich medium like 2xTY or 2xLB)
 * 2) seal plate with gas-transmissible adhesive tape
 * 3) grow over night with vigorous shaking (700 r.p.m.) at 37°C

Contact

 * Raik

or instead, discuss this protocol.