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I just want to clone one paticular immune gene..we have cDNA sequence.. so by means of exon. i design my Primer. bt i am not getting any bands .. finally i got band but i cant able to reproduce that band again. i need to clone that particular genomic sequence of that gene.. i extracted DNA .. and by nano drop..i used 50ng to 100ng of template Dna..is that i need to partial digest genomic Dna. and plz tell your valuable info..for me.
I used 3- 4 min extension time.. denatuartion 1 min i used.. anealling .i used 55 where i got band twice..
Last edited by firstname.lastname@example.org; 07-06-2010 at 04:24 AM.
Re: Genomic PCR
The most common problem with genomic PCR using primers designed from cDNA is that the primer may span a region where there is an intron in the genomic sequence, so it can't anneal. I would redesign my primers. The one band you got may have simply been contamination. Did you do a no template control? Also, with genomic DNA I would use 0.5-1 ug of DNA per PCR.
I have attached the following link which has been very helpful for me.
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|genomic , pcr|
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