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Promoter PCR primer set problem

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Promoter PCR primer set problem

Postby bravebeaker » Tue Jul 09, 2013 3:05 am

Hi all,
Recently I have designed the following primers to amplify a part of the promoter region of my gene of interest.

fw: 5'- gggg CCGCGG TG AGC AAG TAT ACC AAC CAT -3' (Sacii) Tm 63.3
rv: 5'- gggg TCTAGA CAA TGT ACA CCA ACA TAT AC -3' (Xbai) Tm 56.5


I am using genomic DNA as template and KOD kit.
Unfortunately I have been unsuccessful in amplifying the target region.

I have tried many annealing temperatures such as 50 up to 60.

Any idea how to solve this problem? If the primers are of no use, how can I redesign them? I need the SacII and XbaI sites.

Thank you a lot.
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Postby JackBean » Tue Jul 09, 2013 9:29 am

The difference of Tms is pretty big, 7°C! You can either try knock-down PCR or try different primers. But remember, that you need to check Tm of only the part which will bind to the gDNA. Why do you have 4 guanosines in the beginning, that seems like way too much. Both your enzymes are fine with only 1 base in the end: https://www.neb.com/tools-and-resources ... -fragments
http://www.biolib.cz/en/main/

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Postby bravebeaker » Wed Jul 10, 2013 2:39 am

Thank you for the help and the link. I am going to give it another try if not I am going to redesign the the Fw primer.
I calculated the Tm by using the 2+4 rule but it seems its not very accurate.
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