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Large fragment amplification failed


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#1 Vaideesh

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Posted 08 February 2011 - 03:30 AM

Hi everyone
I'm trying to amplify a 3.5kb gene. I divided it into two parts 1.5kb and 2kb amplification. Both the amplifications worked. But when I'm trying to amplify it fully it's showing nothing but the primer dimers. In one or two cases I got smearing.I tried various temperatures.The details are as follows
Sense primer-TTCCTTGTTTTTCATATTGTGT ;Tm=56C
Anti-sense primer-TGGAGAGTTATGCCCTATTT ; Tm=58C
I also tried a gradient from 58.3C-59.1C but didn't get any result. Tried varying the primer concentration but this was also futile.
It would be of great help if you can suggest me what parameters can I change and the appropriate values for amplifying this large fragment.
Expecting a reply from you molecular biologists.

#2 phage434

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Posted 08 February 2011 - 05:08 AM

Annealing is effective around 5 degrees below Tm. Try your pcr with an annealing of 53C. The range of your gradient was very small. Usually I run 50-62. I would have tried for a longer primer with the low GC you have. Also, I would have designed it so that there was a G or C at the 3' end of the primer. In my opinion, people take the calculated Tm values far too seriously. I assume you lengthened the extension time for the longer fragment. Finally, if the organism has low GC, then the extentsion TEMPERATURE can be too high. You could try an extension at 64 instead of 72, lengthening the extension time.

#3 Maddie

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Posted 08 February 2011 - 08:21 AM

I agree with phage. Your forward primer is really low in G & C and I am surprised you got 56 as a Tm.
For oligos longer than 14bp, you could use this formula:
Tm = 64.9°C + 41°C x (number of G’s and C’s in the primer – 16.4)/N

In your case, this gives an annealing temperature of 46°C (but I would add a few degrees to that).
See:http://www.promega.com/biomath/calc11.htm

Have you tried 48 or 50°C?
Can you redesign this primer to get a more balanced ratio between AT and GC?

Edited by Maddie, 08 February 2011 - 09:09 AM.

Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein

#4 Ameya P

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Posted 08 February 2011 - 10:39 PM

Your annealing temperature must come down to get some product. Could you let us know what extension time are us you using. With a normal Taq, it has to be >3 mins to get a product that long.

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