I used 2X Phusion Master Mix during my PCR reaction and after that I run the PCR product (10ul).
I can observe a faint band but I do not why it looks like degradation. I attached a picture to ask what could be the reason of this big smear on the gel?
Thanks a lot for your help.
Mary
Too high DNA template concentration. Repeat with around 20-30 ng DNA in your 25 ul PCR reaction.
Hello!
The smearing of your sample could be due to degradation of the product or excess template. In your case, since it is a PCR reaction, I do not think degradation would be the issue. Do check your template (requires dilution) and dNTP stocks. Use molecular biology grade water in your PCR mix and repeat the gel. Hope this helps!
The smear is due to the degradation of the product or excessive heat generation during electrophoresis.
2. Another reason is contamination in the running buffer.
3. Contamination in PCR reagents.
Please check all these one by one.
All the best.
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