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Genomic PCR

Genomic PCR - PCR - Polymerase Chain Reaction Forum

Genomic PCR - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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Old 07-06-2010, 04:22 AM
Pipette Filler
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Default Genomic PCR



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 vinusiva@gmail.com; 07-06-2010 at 04:24 AM.
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Old 07-06-2010, 09:37 AM
Pipette Filler
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Default 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.
[Only registered users see links. ]

Good luck!
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The Following User Says Thank You to cat1082 For This Useful Post:
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