I am doing over expression of an endogenous target gene in a CHO cell line. When I did PCR (whole length of the gene) to verify the existence of my target gene, I could only see the gel band when the plasmid was used as the template but not the cDNA or the genome. This gene has no introns. And I could see the GFP under the fluorescent microscope which was coded in the plasmid. WB of my target protein also gave the positive results. So what is the problem of my PCR?
Hi there,
Just a silly question: the pair of primers you are using are specific for the coding sequence (ie. not specific for the plasmid upstream and donwstream of the cloning region)?
If you are doing a plasmid preparation then amplifying the product you must remember that the template copy number is very large....eg one template sequence every plasmid genome size ( 5kb perhaps) but the genomic target is one copy evry genome size so it may just be that you are adding much more template in the working plasmid target pcr
There are 3 options l could suggest based above answers
1. Blast the primer pair and check it's specific for the region in the gene or for the plasmid.
2. With the cDNA one needs to standardize the concentration to be used in the reaction, also the quality of the cDNA ( could be checked with any other other primer pair like any house keeping gene)and if there is amplification then quality of cDNA should be fine.
3. Genomic DNA concentration also needs to be standardized in the PCR.
Thanks for all the above suggestions. I did blast my primers on both the plasmid and the genome to verify that they are specific to my target gene before I ran my PCR. And one thing I didnot mention is that I used another pair of primers targeted to the middle part of the gene (half length), the gel band could be seen clearly which means the copy number in the cDNA or genome might not influence the PCR result that much, am I right? If yes, is there any possibility to give the no band result for the whole length experiment? Vanita Malekar Paul Rutland Dominique Liger
If you have an inner primer set are the inner primers usable to run the pcrs with all of the primer combinations. ie outerF-innerR, outer r-innerF as well as outerf-outerR to get a product showing that all of the sequence is amplifiable or,at least hinting at where the problem lies
Hi, Anita Ma,
1) as Paul suggested, there may be a problem of sensitivity. You could check this by titrating down the amount of plasmid construct that you are using as positive control for PCR, and see. How much genomic DNA or cDNA are you using for PCR? How long is the PCR product(s) that you expect?
2) Have you checked that there are no SNPs in your cell line in the sequences where your primers should anneal? Again, by trying different primer combinations you should get a product. Try small amplicons first.
Good luck!
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