PCR Screening for Targeted Stem Cell Colonies in 96 Well Plates

  • Use multiple channel pipet to pool DNA samples from each column, 10 µl from each well (one mixed sample contains DNA from eight wells). Mix thoroughly.
  • Perform nested PCR.


Stock Concentration

Amount (µl)

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

94°C 3 minutes  
94°C 30 seconds  
55°C 30 seconds  
72°C 3 minutes (1 minute for 1kb) 35 cycles
72°C 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).