Hi guys,
I have been trying to amplify fragments of DNA(different sizes from 650bp to 1400bp) from separate plasmid DNA. I had to design primers that contain overhangs manually which aiming to amplify the complete DNA sequence. However, when I ever try to conduct PCR reaction, gel image shows that amplification at all genes occurs at 500bp.
I've done so many runs with modification each time and this includes:
- temperature gradient
- several DNA conc.
- fresh H2O and new primers
- changed taq polymerase
- several number of cycles
- changed primer conc.
- changed PCR reaction from conventional PCR to touchdown PCR.
with all of these changes, I still get 500bp at all genes.
could you help me please?
do you always run a no template control in your pcr amplifications and does this band appear in the no template control?
Do the primers BLAST only to the correct gene sequences?
Dear Mo,
I agree with Paul Rutland do you have a negative control in your PCR and how does it looks like? you should have no band at all since you do not have any telpmate ..
Also, about the primers how good they are ? I mean do they match the correct gene sequences? again as Paul said did you do a BLAST to check them?
From the photo you send looks like you have a band of about 50 bp in lanes 5 and 6 and while in the lanes 11 and 12 the most intense band is at about 200 bp along with a series of other faint bands (all these sizes taken from the marker you put in gel...). I see no band at 500 bp as you said in the text !!
Could you put the gel image which shows your PCR result those you are talking about so we can help you better and understand the situation.
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