Hello! I want to know the genetic variability of C. acutatum collected from Korea
So, I am working on the AFLP analysis of C. acutatum.
first, I conducted the pre-amplification step and amplification step of AFLP by the conventional PCR method.
( 95: 3m, 95: 20s - 55:20s - 72:1m (25cycle), 72: 5m )
But, after the silver staining, I got non-specific and non reproducible band patterns..
So, Secondly, I conducted the amplification step of AFLP by the touch-down
PCR method instead of the conventional PCR method.
= 95: 3m, 95: 20s - 65:20s - 72:1m (10cycle, In each of the following 10cycles, the annealing temperature was reduced by 1℃). And the last 25 cycles were carried out at an annealing temperature of 56℃.
However, I got smear band patterns.
I have attached the figure of my results.
the smear band patterns are normal?? or are there any problem of my protocol?
have you checked your primers to make sure that one primer is not in a common repeat sequence so that 1 primer in different orientation may be producing smears. It might be worth a control sample with one primer next time to check if one primer is a problem and can produce this effect. You might consider designing a new primer set. You should be getting bands.
Hi Jong
In my experience with AFLPs, DNA quality is very important, because if DNA is not clean it will have digestion problems.... therefore if the digestions is not uniform and complete, you will have reproducibility problems. The photo of the touch down for me shows uncomplete digestions, because you can see strong bands of hight weight in some samples near the well. Have you checked the complete digestion of your DNA?
Follow the instruction manual provided with the kit you are using.
But DNA quality is very important in AFLP.
Dear Jong, For Pre-selsctive amplificatio you expect the smear but for the selective amplification, if properly optimised, the bands should be visible by silver staining. (Have acheived very good bands even on 2% Agarose). Follow the protocol well and the dilutions of products to be used in the proceeding steps affects the quality too.Best wishes.
DNA quality and complete digestion of the DNA are essential for good AFLP pattern. You need to concentrate on the final amplification pattern. If you find much smear in the preamplification you may slightly increase the annealing temp.
Did you check the quality and quantity of DNA before you pre-amplify. As mentioned by above experts, for AFLP, the quality of DNA is must. The primer's pair combination is also important. I usually pre-screen the primer pair combinations.
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