Hi Everyone,
I performed a PCR reaction. On agarose gel, the band corresponds to 500 bp with respect to the marker. The gene size is 866 bp. Please give any suggestion for this problem.
Markers are only an approximation. There're few things that can affect your run such as your sequence, for example, depending on your % of A and G, since those nucleotides are "heavier". If you want to be sure that your band is the right one I will send it for sequencings... It's quite cheap nowadays :)
If you used Gelred to stain your PCR product, you should dilute your PCR product 20-30 times before loading on the agarose gel.
The product will not match exactly the marker, but should be close. 366 bp difference is too much. Check the sequence of your primers. Sequence the product as mentioned above.
I agree with the above suggestions with confirming your amplified gene product sequence as that is the most probable cause of such a huge difference in bp. If so and if you have designed these primers, please cross-check to see if they will bind to your regions of interest only on a well-curated database, before attempting another PCR. It will save a lot of reagents and time! :)
As Maria Belen says, I think there is much difference between what you see and what you want, I think the problem is an amplification that does not correspond with the product you are looking for, try to raise the annealing temperature of your reaction
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