PCR an insert in a vector with wrong product size - I can not amplify my fragment with a normal PCR (Mar/23/2005 )
I have a plasmid from a cDNA library. The cDNA of my interested gene is somewhere inside this vector. I desigend a couple of primers fo my target gene and did PCR, but everey time I get a band with wrong size ( may target must be 1kb but the PCR product is 5.5kb), I did blast with my primers and also checked it for my vector , every thing is OK, the primers don't recognize any sequence in the vector except my target!!!
Could you help me
....but everey time I get a band with wrong size ( may target must be 1kb but the PCR product is 5.5kb)...
how big is the whole plasmid? I'm guessing its about 6.5 kB. Am I right? Make sure your primers are pointing in the right direction to amplify your 1 kb fragment, I'm guessing the primers are all pointing out, amplifying everything else EXCEPT the fragment you want.
My vector withouth insert is 4900bp but I dont know exactly the size of insert, Its from a cDNA library, I bought it from a company and they sure that the insert include the cDNA of my gene.
I even checked the primers with different sofware, (primer3 and..) The direction of Primers also is correct.
I can't think of any other reason why it would be like that, from what you've said so far.
You may want to try to digest your plasmid so that you release your insert, purify that band, and do a PCR on that.
I know this sounds stupid but make sure you're working on the right plasmid. coz I don't see why you shouldn't get your right sized bands.
Now I'm running another PCR, if this PCR also will be tha same, May be I cut my insert, but first I should find the restriction enzymes that they used for cloning.
did you do a pcr on your vector alone (if possible)?
I remember someone had the same problem before in this forum. It could be that the extension of PCR from one primer run all the way aound the vector till it meets the another primer again. So the total size will be
vector size + (insert)^2
I think It's just your template that you see on the gel
try again PCR with negative control,, it's with the same template but without polymerase,, you should see again this band on the gel for your negative control
good control, arianomics.
But, really, if you can see your template in the gel, than there's too much template. PCR works optimally with minute amounts of template.
keep us posted.