non specific amplification in clone fragment using primer aganist vector

[WS]

Dear Muhammad,

Possible reasons: Problem with the insert or problem with the primers.

Do you have any unrelated fragment in the same vector where you know
that the insert is ok?
Did you try a gradient PCR?
Do you get the same band when you employ just 1 primer in the rxn?
Can you cut out the insert and check its length?

Wo


muhammad yasir wrote:
> i have constructed 16s rRNA library in topo TA cloning vectore of invetroge and desigen primer for vector region near to the cloning site. but in the screening i am getting one light band that is extactly double (3kb) of the clone fragment (1.5Kb). i shall be very glad to know from some one, why i am getting this non specific amplification and how to solve this problem.