How to re-amplify the DDRT-PCR band?

nhwu at PUBLIC3.BTA.NET.CN nhwu at PUBLIC3.BTA.NET.CN
Thu Mar 13 21:51:33 EST 1997


Dear netters,

I pleased my boss by good result of Differential Display. But I cannot 
re-amplify the band I interest in.  I used the following protocol:
  *cut the band from the filter paper; use 100ul water soak it for 30 minutes,  
   then boil for 15 minutes. Recover the water.
  *in a 0.5 ml Eppendorf, add 
	31.6ul		eluted DNA
	5ul		10x PCR Buffer
	5ul		1.5mM MgCl2
	4ul		2.5mM dNTPs
	2ul		arbitrary primer(20pmol)
	2ul 		dT11VV primer(40pmol)
	0.4ul		Taq(2U)
   *Then 
	94C	5 minutes	1 Cycle
	    94C	  1 minute	\
	    42C   1.5 minutes   |  40 Cycles
	    72C   1.5 minutes	/
	72C	10 minutes	1 Cycle

It seems right, but I got nothing. Someone told me using 5ul of first round 
amplification for second round PCR, and I just got smears.

I am sure that there must be some details I ignored, but I don't know what it 
was. Could someone there can give me some advice? I will greatly appreciate.

Xiao-yu Liu

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