We use 3 ul BigDye dGTP, 3 ul of 3.3X Buffer(Tris/MgCl2), 3 ul 100 ng/ul template, and 1ul of 3.2
pmol/ul primer. Nothing special there.
Maybe the PCR profile makes a difference. Here is the one we use.
95* 5 minutes
94* 10 seconds
55* 5 seconds
65* 2 minutes
(cycle 30 times)
65* 5 minutes
This is 5 degrees hotter annealing and extension times than recommended in the ABI guide. I can't
find an example of a long string of C, but with 70% AT or GC we have good results.
I hope this helps!
Sheryl Christofferson
OMRF DNA Sequencing Facility
Oklahoma City, OK
www.omrf.ouhsc.edu/OMRF/Core/DNASEQ/
HOSSEINI@gmm.gen.emory.edu wrote:
> Folks: I am sorry for incomplete posting. I think our email system was not functioning properly.
> So I will try again:
>
> Sheryl:
>
> We used the ABI's dGTP kit to sequence several DNA fragments with high
> GC content (for example a row of 12 Cs) and we got the same results as the conventional BigDye
> kit.
> I tried several samples and didn't see any difference. I saw your data and they were great.
> Is there any particular way that this kit should be handled in order to read through GC content?
>
> Thanks
>
> Seyed Hosseini
>
> Seyed H. Hosseini, Ph.D.
>
> DNA sequencing lab
> 1462 Clifton Rd. Rm#416
> Center for Molecular Medicine
> Emory University
> Atlanta, GA 30322
> Tel # 404-727-1353
> Fax # 404-727-8367
> hosseini@gmm.gen.emory.edu