Large fragments from PCR. - problems getting 3.5kb from genomic DNA (Nov/02/2006 )

Hey all,

I've been trying to isolate a promoter from zebrafish genomic DNA and have had no luck. Its a big chunk, 3.5 kb, so that may be the problem. My primers anneal at 62.44 degrees and the program I've been running on the thermocycler looks something like this:

94- 5:00
94- 1:00
54- 1:00
72- 5:00
repeat 35 cycles
72- 15:00
4- for ever

When I run my product out on a gel I get no bands or maybe one non-specific band and usually a genomic smear in the background. I even tried re-amping from the last product with high-fidelity Taq and still got nothing. Does anyone have any suggestions or know of something that I'm doing wrong. I really need to figure this out soon. Thanks.


Yelena

P.S. the attachment is of the last PCR which was the re-amp with the high fidelity taq, wells 6 and 7 were positive controls but there is some non-specific stuff in there. The positive control band is the one that's areound 600-630 bp.

is the product you expect to see 630 bp or 3.5 kb?
is the positive control from genomic DNA or from a plasmid?

i would think that the primers aren't annealing. have you tried using a gradient PCR?

more questions than answers, sorry.
if it is the 3.5kb product you want, perhaps you could invest in a polymerase that is specific for long range PCR.

V

What polymerase are you using? As vetticus3 has mentioned you need a high fidelity polymerase. I like KOD hifi.

How is the quality of your genomic DNA? Is it degraded in anyway? Is it free from proteins and other junk? Can you do a PCR reaction on the genomic DNA you have, perhaps using primers that you know work?

How is the region that you are amplfiying? Is it rich in dinucleotide repeats? Some areas are just bad primer binding areas. If worse comes to worse you may have to redesign the primers

What is your PCR mix like? Perhaps you can -
add more MgCl
add DMSO
add BSA
add glycerol
-into the PCR mix.

Try touch down PCR? Maybe that will help?

I am surprised that there is such a big difference between the primer's melting temperature and annealing temperature.

Hi :

May be your Denaturing time is a bit long，Try 94C 30Sec.

I too had a similar situation when trying to amplify a gene from a AT rich genome. I used to get a non-specific band around 750bp and a smear following it while my expected amplicon was 2 kb. Decreasing the extension temp from 72 to 60 ( as recommended for AT rich genomes)helped me get a clear band at 2 kb.