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PCR Screening for Targeted ES Colonies in 96 Well Plates

  • Use multiple channel pipet to pool DNA samples from each column, 10 l from each well. (one mixed sample contains DNA from eight wells). Mix thoroughly.
  • Perform nested PCR.
Stock Concentration
Amount (l)
H2O   10.7
PCR Buffer 10 X 1.5
dNTP 2.5 mM 1.2
MgCl2 50 mM 0.45 
Primers 25 uM 0.2 each
Taq 5U/ul 0.25
Total Volume   14.5
DNA Template   0.5

PCR Cycles
94C 3 minutes  
94C 30 seconds  
55C 30 seconds  
72C 3 minutes (1 minute for 1kb) 35 cycles
72C 10 minutes  

Use the same nested PCR to screen individual well in the row that the pooled sample shows positive.

We use Platinum Taq (Invitrogen catalog number 10966) for PCR product < 3.5 kb. For PCR product longer than 3.5 kb, we use Elongase (Invitrogen catalog number 10480-010).