long range PCR problem - unable to amplify 12 kb fragment (Jun/27/2005 )

Hi,

I'm having problems with amplifying 9 kb and 12 kb genomic DNA fragments. I have tried two different enzyme mixes (Roche Expand Long Template PCR System and Eppendorf TripleMaster PCR System), different primer sets designed specifically for long range PCR, various cycling programs,... without success. All I get is a high weight smear and no bands. I am running out of ideas.

Does anyone have suggestions?

-moleculan-

Try using BD Advantage Genomic Polymerase

-axisy-

Hi moleculan,
What DNA extraction method that you are using?
What is your amount of DNA extracted?
How is the purity?
How much MgCl2 that you use in your PCR reaction?

regards

-Hadrian-

Try adding betaine to your buffer using a concentration gradient from 100mM to 1.3M. Also have you tried manually hot starting your reactions?

Daniel

DNA sequencing software

-Daniel Tillett-

any possibility to check the integrity of your DNA (f.i. shoter PCR on the same template?)

-vairus-