Edited by georgepatra, 24 March 2005 - 12:17 AM.
3 bands on RT-PCR
Posted 21 March 2005 - 06:23 AM
Posted 22 March 2005 - 02:32 AM
I am not sure what you mean by 'zone', but if you are talking about three bands on the gel, I would think some of them might be non-specific products.
Either that, or your protein has some splice forms that you pick up in your RT-PCR
Posted 24 March 2005 - 01:36 PM
Posted 26 March 2005 - 01:30 AM
There are two posibilities that can make you RT-PCT generate more than 1 band.
First, your primer not specific enough. Not so much during PCR but more important during your RT step. As we know, RT step has to be carry out at lower temperature (~50oC). This might be how the unspecific band come about.
Secondly, some of your RNA had degraded into smaller fragment. Thus the small fragment actually serve a "primer" during PCR. So now you got "extra primer" in your solution. That why you can get multiple band.
Posted 28 March 2005 - 06:19 AM
2. Then try to increase the annealing temperature in your PCR step.