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.
Ji Hoon Ahn, PhD
PBIO-A (PDMG: Lab of Plant Developmental Molecular Genetics)
College of Life Sciences and Biotechnology,
Anam dong 5 ga, Seongbuk-Gu,
Email : jahn at korea.ac.kr
Homepage : http://biotech.korea.ac.kr/~PDMG