Large fragment amplification failed
Posted 08 February 2011 - 03:30 AM
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.
Posted 08 February 2011 - 05:08 AM
Posted 08 February 2011 - 08:21 AM
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).
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.
Posted 08 February 2011 - 10:39 PM
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