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No PCR product with verified primers on templates - Where is the bug???? (Dec/11/2007 )

I have a strange problem with a PCR.

I have 2 sequence verified templates A and B. B is a truncated version of A lacking the first 300 bp resulting in a 500 bp gene.

I have also two primer pairs:
A_fwd with A+B_rev resulting in a product
B_fwd with A+B_rev not giving any product

I have also a second rev primer which differs from the other one in introducing a 6xHIS sequence after the coding sequence.
Thereby resulting in the primer pairs:

A_fwd with A+B_HIS_rev not giving any product
B_fwd with A+B_HIS_rev resulting in a product

That means each primer has at least once worked with the template sequence (since B is identical to the last 500 bp of A).

I have checked selfannealing or unspecific priming of the primers on the template vectors via pDRAW32. There was no such thing (and I do not seee any unwanted bands in the PCR).

I have also ran temperature gradients (from 45 to 72 °C), used different enzymes (taq, pfu, Phusion), hot start, primer excess (10x more primer). I even changed the template to a different vector (pET26 to pCR blunt)
Nothing has worked so far.

The primers all have a 19-21 bp region to prime the template, a 12 bp overhang for RE recognition sites (plus the 6xHIS for the HIS primer), they end with either G or C and have an calculated TM of 68 to 72 °C (4xGC/2xTA).

Do you have any suggestions what could be the reason for not getting any product in some cases? What can I additionally do to get one?


Just need a clarification, what is your template exactly and what concentration...


QUOTE (beccaf22 @ Dec 11 2007, 09:12 PM)
Just need a clarification, what is your template exactly and what concentration...

I used 50 or 100 ng of template in a 50 µl reaction.

But my problem is solved meanwhile. :-)
Using diffenernt annealing temps for the first 5 cycles of the PCR reaction (54°C) and then switching to 65°C for the next cycles worked.