A problem on TAIL-PCR

[Ji Hoon Ahn]

Dear netters,

Recently we have had a terrible problem doing TAIL-PCR to find 
insertion sites from an activation tagging population in a large 
scale. The thing is that bands are all smeared, we couldn't find any 
bands from the experiment. One weird thing is that at first it worked 
but as time goes by smeared pattern appears.

These are what we're using
Platinum Taq (or HotStart Taq from Qiagen)
Degenerate primer sets (AD1, AD2, AD3 from GenSet)
Quick and dirty genomic DNA prep using blue pestles
3 rounds of PCR as suggested in the original protocol (Liu et al., Plant J)
MJR PTC-200 machine

Does anyone have a reliable protocol to do TAIL-PCR or similar 
experiment to find insertion sites?

I'd appreciate if you share your tips on your TAIL-PCR with me.

Thanks very much in advance.

Best regards,

Ji Hoon

--
Ji Hoon Ahn, PhD
Assistant Professor
PBIO-A (PDMG: Lab of Plant Developmental Molecular Genetics)
College of Life Sciences and  Biotechnology,
Korea University
Anam dong 5 ga, Seongbuk-Gu,
Seoul, 136-701
South Korea

Phone: 82-2-3290-3451
Fax: 82-2-927-9028
Email : jahn at korea.ac.kr   
Homepage : http://biotech.korea.ac.kr/~PDMG