AFLP double bands in bad profiles
Posted 23 January 2005 - 10:18 AM
These last weeks several people in our lab got AFLP profiles with less bands than usual, but all bands were double (2-3) bp between the peaks, less for shorter fragments and more for longer. It is not due to bad template DNA (replicates from the same isolation ded not have the problem).
Any idea about the cause or what to do?
Posted 25 January 2005 - 06:33 PM
Actually I can't get exactly what you mean by "all bands were double (2-3) bp between the peaks". Is it something like ghost band?
Anyway for the less shorter fragments and more longer one, one possible reason may be the incomplete digestion of your DNA template. Did you make any changes in the digestion and ligation process as before? Is the digestion time long enough (have to check according to the restriction enyzme you used)? Sometime if there is inhibitors (like polysaccaride) in the DNA extracts, they will affect the digestion. Is the DNA template you used from new organsims? Maybe you can check.
Posted 26 January 2005 - 12:23 PM
Yes, you can call it ghost bands. Many bands were missing, but instead there were a few peaks, always two quite close together. I did not find out what it was yet, but set up a row of reactions now to find out. I exclude problems with the template, because I had some replicates from a plate which worked fine a week before. Also the the reaction times were fine earlier. I also thought about inhibitors. Something might have been wrong with the salt or BSA concentration. I will post the reason when I find it...