GTTF/96 well PCR

		Protocols


Services & Fees Mouse Colony Information Links Contact Us Home	PCR Screen Targeted ES Cell Colonies in 96-well Plates - Use multiple channel pipet to pool DNA samples from each column, 10 µl from each well. (1 mixed sample contains DNA from 8 wells). Mix thoroughly. - Perform nested PCR.

PCR Mix:
		Stock concentration	Amount (ul)
	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 min
	94°C	30 sec
	55°C	30 sec
	72°C	3 min (1 min for 1kb)	35 cycles
	72°C	10 min

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

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