Hello, I am facing problems with my PCR. My gel turned back smeared. Can anyone help? Before, it was working fine when I used the same set of primers and PCR mix. What may cause the problem now??? Attached is the picture of the gel
it could be over amplification caused by post pcr contamination of one of your reagents. Do you also get this effect in your negative control ( no template control) ?
I've seen that from either dirty gel rig parts, loading too much product, or old running buffer.
Wash out the gel box, gel combs, casting tray, etc.
Your ladder is a bit "wobbly". Make sure you aren't running at too high a voltage.
Make a FRESH batch of running buffer and make sure your gel is completely solid before you take out the combs.
First thing to do, test your genomic DNA as it might to be fragmented.
Second, give the gel enough time to solidify (use clean TBE buffer 1X).
Third, use good TBE buffer and adjust your voltage and ampere (100v and 60mA) by using 0.5X. 0.6X or 0.7X TBE running buffer.
Finally, from the pic attached, it seems that you have a non-specific band so adjust the PCR conditions (e.g. raise the annealing temp. by 1Co )
Good Luck
Hello
I think several things are happening. It seems you are running your gel too fast, I think your samples are running faster than they should so you see that smear, also your marker seems to be fragmenting in a way that is not smooth, if that makes sense.
I am also agree with Hassan Darwish, you seem to have several bands in your samples. This could be due to the sample itself (is it too old after the PCR? Did you freeze it at -20 at least?) or maybe due to the primers, do you have some dimerization in your PCR?
Thanks all for your answers..I will try them out and will let you know if anything changed.
Thanks again
Depend on what type of gene and its size your are amplifying.
simply there are no amplifcation in all of your pcr product.
also worth to add quality and house keeping controls such as beta-actin or 18s.
This way you can check your priners and Taq polymerase quality.
Hope you find the answer.
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