Lunatic is available upon request. The ongoing ABRF DNA Sequencing Research
Committee 1999 Survey is still open for sample submissions. Please submit your
data before June 1st so that it can be included in the next report of results.
The study has two parts. The first part involves a study with a standard pGEM
template. Information on how to submit data for this part of the study is
posted at http://mbcf.dfci.harvard.edu/dsrc.html. The second part of the study
involves the results of sequencing challenging templates, called Lunatic and
Sasha. Instructions on how to request samples and submit data can be found at
the Difficult Template Study web site at http://brcweb.bio.cornell.edu/dsrc99.
Thanks for your interest in this study.
- The ABRF DNA Sequencing Research Committee
At 09:09 AM 5/17/99 -0500, Sheryl Christofferson wrote:
>
>
>"m.p.woodland" wrote:
>
>> 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
>
___________________________________________________________
George Grills
Director
DNA Sequencing, Oligonucleotide, and GeneChip Microarray Facilities
Albert Einstein College of Medicine
713 Ullmann Building
1300 Morris Park Avenue
Bronx, New York 10461-1602
Tel: (718) 430-2657
Fax: (718) 430-8778
E-mail: grills@aecom.yu.edu
DNA Sequencing: http://leper1.ca.aecom.yu.edu/dnacore
Oligonucleotide: http://sequence.aecom.yu.edu/oligo
___________________________________________________________