PrbbBB:colony pcr v1
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).
- 96-well PCR plate
- AmpliTaq DNA Polymerase 5 U/µl (Roche)
- AmpliTaq Buffer 10 x
- dNTP mix 10mM each nucleotide
- LB "backup" plates (with Ampicilin), one for each assembly reaction
I PCR reaction
- prepare 96-well "Lysis" plate:
- multi-dispense 50 µl H2O into each well
- pick 8 colonies of one assembly into the wells of one column (like A1-H1 or A2-H2)
- 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")
- proceed to next column for next assembly
- prepare PCR plate:
- multidispense 10 µl PCR mastermix into each well
- copy 1 µl per well from lysis plate to PCR plate (using multi-channel pipette)
- seal PCR plate with adhesive tape
- run PCR program
- put backup plates @ 37 °C
II Agarose Gel
- prepare a large 0.7 - 1.5 % Agarose gel (depending on fragment size)
- multi-dispense 2 µl loading buffer into each well of the PCR plate
- load gel with multi-channel pipette
- run, analyze, enjoy!
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
raik: no comment
or instead, discuss this protocol.