> M.P.Woodland BSc(HONS),Ph.D
> DNA Sequencing Facility Manager,
> Dept.of Biochemistry,
> Imperial College of Science,Technology & Medicine,
> Exhibition Road, South Kensington,
> London SW7 2AZ
> Phone:- (44) 0171 589 5307
> Email:- m.p.woodland@ic.ac.uk
>
> ----------
> >From: Sheryl Christofferson <sherylc@omrf.ouhsc.edu>
> >To: Recipients of ABRF List <abrf@aecom.yu.edu>
> >Subject: Re: GC content of DNA.
> >Date: Fri, May 14, 1999, 7:47 pm
> >
>
> Hi Sheryl,
>
> Your comments on the dGTP kit sound interesting:-
>
> A couple of queries - I have used the std Big Dye kit which has
> a 4min extension time is the 2 min extension time part of the
> dGTP protocol kit protocol or did you find by experimentation that
> this was sufficient ?
>
I have heard that a rule of thumb for PCR is to allow 1 minute extension for every
1000 bases of product. Since the max the 377 XL can give (legibly), it seemed
reasonable to reduce the extension time just to make the prep part go faster.
>
> Why a single final hold at 65 for 5mins ?
Force of Habit.
>
>
> How much do you load on the gel - I used to resuspend a 10ul
> reaction in 2ul and shoot the lot with the std BigDye kit ?
Resuspend 10ul in 3ul of formamide dye and load 1 ul.
>
>
> I havent seen your results but what do you consider a good
> read/accuracy for std ds GC rich ?
I cannot vouch for accuracy since, as a Core Facility, I do not analyze the DNA. But
it looks good out to 750-800 bases.
>
>
> Do you know of a 'std" GC rich template ?
The ABRF research group had one called "Lunatic" that they were testing. Maybe someone
could provide some?? I discovered mine during routine sequencing.
Hope this helps!
Sheryl Christofferson
OMRF DNA Research Facility
Oklahoma City, OK
www.omrf.ouhsc.edu/OMRF/Core/DNASEQ/
sherylc@omrf.ouhsc.edu
>
>
> No rush but if you could find the time we can continue in the
> Spirit of the NATO alliance I know keep politics out of DNA
> sequencing.
>
> Regards,
>
> Marc
>
> >Seyed:
> >
> >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/
> >
> >
-- Sheryl Christofferson
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
OMRF DNA Sequencing Facility
Oklahoma City, Oklahoma
(405) 271-7393
www.omrf.ouhsc.edu/OMRF/Core/DNASEQ/
sherylc@omrf.ouhsc.edu