PrbbBB:colony pcr v1
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==Materials== | ==Materials== | ||
| - | *96-well PCR plate | + | * 2 96-well PCR plate |
| - | *AmpliTaq DNA Polymerase 5 U/µl (Roche) | + | * AmpliTaq DNA Polymerase 5 U/µl (Roche) |
| - | *AmpliTaq Buffer 10 x | + | * AmpliTaq Buffer 10 x |
| - | *dNTP mix 10mM each nucleotide | + | * dNTP mix 10mM each nucleotide |
| - | *ddH2O | + | * ddH2O |
| - | *primers: | + | * primers: |
| - | **[http://partsregistry.org/wiki/index.php/Part:BBa_G00100 BBa_G00100 (VF2)] [http://brickit.crg.es/registry/biobrick/5/ CRG-internal registry] | + | ** [http://partsregistry.org/wiki/index.php/Part:BBa_G00100 BBa_G00100 (VF2)] [http://brickit.crg.es/registry/biobrick/5/ CRG-internal registry] |
| - | **[http://partsregistry.org/wiki/index.php/Part:BBa_G00101 BBa_G00101 (VR)] [http://brickit.crg.es/registry/biobrick/6/ CRG-internal registry] | + | ** [http://partsregistry.org/wiki/index.php/Part:BBa_G00101 BBa_G00101 (VR)] [http://brickit.crg.es/registry/biobrick/6/ CRG-internal registry] |
* LB "backup" plates (with Ampicilin), one for each assembly reaction | * LB "backup" plates (with Ampicilin), one for each assembly reaction | ||
Revision as of 10:02, 26 February 2009
Contents |
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
- 2 96-well PCR plate
- AmpliTaq DNA Polymerase 5 U/µl (Roche)
- AmpliTaq Buffer 10 x
- dNTP mix 10mM each nucleotide
- ddH2O
- primers:
- LB "backup" plates (with Ampicilin), one for each assembly reaction
Procedure
I PCR reaction
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- 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!
Notes
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
raik: no comment
References
Contact
or instead, discuss this protocol.
