What kind of polymerase did you use ? For such amplification, I guess you should use a special enzyme... maybe Phusion® High-Fidelity DNA Polymerase (BioLabs). As far as I remember, Clontech also have special enzyme : Advantage® 2
PCR Enzyme (TITANIUM™ Taq DNA Polymerase)
This enzyme seems to be good for long fragment amplification... what about the extension step you use ? I guess you perform a 7 - 8 minutes extension step, and 30 to 40 cycles right ?... if it is a problem of specificity of your primers, you can try a touchdown PCR.
You also have to be sure that your primers are well designed. I guess that a 7 kb fragment is a genomic fragment, you can test your primers on cDNA (fragment should be shorter and easier toà amplify)
can u tell me the Tm of your primer (which come with data sheet) and annealing temp you set in PCR, also inform me the Taq u are using
thank you very much for reply I will check it when I will back to lab .. Regards
it is seen that Taq pol from phusion is most sought for amplification of large size molecule. It is high fidelity pol from New England Biolabs. You can try this
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