PCR screen for transformants (DSG 6/13/03 - currently being tested).  

In the past, this has been inconsistent.  I think it might be best to set up the screen so that both the parental clone and the desired clone produce products but the products should be different sizes.  This might help reduce false positives formed when a miniscule amount of contamination causes parental clones lacking inserts to produce a positive PCR signal.  

Begin by picking single colonies, streaking them on a second plate and finally swizzling the innoculation device in 10 ul of water in a PCR tube to disperse some cells for PCR. Include a colony containing DNA with no insert as a control. Prepare a cocktail consisting of the following for each PCR reaction (multiply volume by the number of samples plus 0.5):

• 10.85 ul water
• 1 ul 10 mM dNTP
• 2.5 ul 10X GeneChoice Buffer
• 0.2 ul (20 pmoles) primer 1
• 0.2 ul (20 pmoles) primer 2
• 0.25 ul GeneChoice Taq

• First cycle: 3' at 94^oC, 1' at annealling temperature, 1' at 72^oC
• 28 cycles: 1' at 94^oC, 1' at annealling temperature, 1' at 72^oC
• Final cycle: 1' at 94^oC, 1' at annealling temperature, 10' at 72^oC