Why I always get smear in the third step of TAIL-PCR? - TAIL-PCR (Nov/10/2004 )

In the first and second step of TAIL-PCR, I can see clearly some bands from 600bp to 2kbp. But at the final step, the PCR product is always smear and can't be isolate by the gel while the DNA marker is ok. My loading buffer is not a problem, I think, because I always use it to load PCR products to the gel.

Hi

It depends for which purpose you need to conduct the 3rd step, because I've read that for nice sequencing two steps are enough.


Moreover, I was wondering if YOU could help me.
I have being tried to recover flanking regions using TAIL-PCR and until now, only unexpected products showed up, meaning that sequences of the transformated plasmid outside of LB-RB region appeared. Another problem is getting no product at all (or I get multiple bands patterns, including negative controls). I tried to use different TAQ polymerase, but results remained the same. I was wondering if you have any ideas or if you encountered similar problems and perhaps have suggestions for me. Do you think that it is possible for all the plasmid to integrate into the fungal DNA?
I would be extremely thankful for any of your ideas or suggestion.

Youlia

I guess the well of the sample goes to diffuse leading to what you said about appearance of negative band

by the way, how does your reaction system form , what volume of each substances?