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PCR with phophorylated primers

PCR Primer

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#1 Ju Hun

Ju Hun

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Posted 14 January 2012 - 04:41 PM

Hello,

I have a trouble in PCR with phophorylated primers.
I used FAM-Forward Primer and Phophorylated reverse primer. (Template < 100bp).
As a Polymerase, Hotstar Taq polymerase is used for our PCR (30 cycles).

My issue is that after PCR (30 cycles),
just FAM Forward Primer and Phophorylated reverse Primer showed the unexpected band
(pretty similar location to the Template DNA+Complementary DNA band) without template and complementary DNA.

Do you know why these bands are existed?
Could you please help me to understand why?

Ju,
Thank you.

Edited by Ju Hun, 14 January 2012 - 04:42 PM.


#2 bob1

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Posted 15 January 2012 - 11:21 AM

It sounds like one of your reagents or tubes/pipettes etc. is contaminated - you should screen each of the reagents for the contaminant by using fresh working stocks individually - of course you can throw out the cheap parts such as the water and 10x buffer, replacing those with new ones.





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