My Forward Primer have 35 nt and Reverse is 32 nt in E.Coli with swing-PCR method and my question is which features could affect like this to smear DNA bonding?
Note: First layer after ladder is Forward Primer of Mutant with Reverse primer of Wild-Type of E.Coli and second layer is opposite of first layer that mean Reverse Primer of Mutant and Forward primer of Wild-Type and other layers is like this pattern.
Thank you
This could be single primer amplification stopping randomly at different lengths. Possibly also you may be amplifying too much genomic dna and what you are seeing is degraded template dna. What does a gel with un amplified dna look like and how much dna template are you using
This could be single primer amplification stopping randomly at different lengths. Possibly also you may be amplifying too much genomic dna and what you are seeing is degraded template dna. What does a gel with un amplified dna look like and how much dna template are you using
First of all, please redesign your sets of primers that should not be more than 25mar. and try the PCR with a low concentration of magnesium chloride, with short amplification time. And the concentration of DNA (template) should also be checked.
Absolutely agreed with dear Paul
Just look at your wells
Full of heavy chain DNA and there's near no amplification
Dear Ali,
I also agree with Paul Rutland. Just looking at your gel I see a lot... of DNA near the well. The gel is not good at all even the marker has a smear.There are some problems with both PCR and gel electrophoresis.
But can you show us the amplified DNA band in a gel? Can you provide more details so you may get better help.
@ Dan Sh
I have a question, you said that you had smears so how did you do purification????
And addressed size????
You must modify and troubleshoot your first PCR. Change and check temp, primers and you name it.
Regards.
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