TTE gels

George S.Grills grills at aecom.yu.edu
Thu Jul 27 11:01:37 EST 2000


Hi Michelle,

There are a number of different DNA sequencing protocols from different
sources that use TTE.  I have seen some that have the loss of resolution
that you describe.  What protocol are you using?  We have developed a TTE
protocol (the protocol Glenis refers to) that consists of using porous
combs, water loading, TTE buffer, and modified run conditions.  Using this
method on an ABI 377, we typically achieve more than 1000 unedited bases at
98% total accuracy and about 900 unedited bases at 100% total accuracy with
standard templates such as pGEM.  In addition, we see significant
improvement in read length with many difficult-to-sequence templates, such
as GC rich templates.  We can get clean reads from the start of the
sequence.  Please let me know if you would like me to send you details of
the protocol. 

- George 


At 11:28 PM 7/26/00 +0100, Glenis Wiebe wrote:
>
>Hi Michelle,
>
>No, we haven't had this problem (knock on wood).  I presume you are seeing
>this with your controls as well?  Sorry I don't have anything helpful to say
>at this point.
>
>Glenis
>
>Biopolymer Facility wrote:
>
>> Hi Glenis,
>> I too have tried the TTE gels, but my results were not as good as yours
>> sound. What I observed was a great loss of resolution within the first
>> 30-50 bases - many overlapping peaks that appear to have migrated oddly and
>> are just kind of jumbled all together, not at all what I typically see.
>> After that 50 bp point the data is very nice. Is this something you have
>> encountered as well?
>>
>> Michelle Detwiler
>>
>> Hi Kev,
>> >
>> >After we tried the TTE gels, we never looked back.  Yes, the results are
>> >that much better. (My thanks to George Grills for getting us started.)
>> >
>> >Here are the basics:
>> >
>> >GEL (5.3% Long Ranger, 6M urea, 1.2X TTE)
>> >21.6 g urea
>> >6.4mL 50% Long Ranger gel solution
>> >7.2mL 10X TTE
>> >final volume 60uL
>> >
>> >30 uL TEMED
>> >300 uL 10% ammonium persulfate
>> >
>> >UPPER BUFFER - 2X TTE (700mL)
>> >140mL 10XTTE
>> >560mL dH2O
>> >
>> >LOWER BUFFER - 1X TTE (900mL)
>> >90mL 10X TTE
>> >810 mL dH2O
>> >
>> >10X TTE (1L)
>> >121.65 g   TAPS (N-tris[hydroxymethyl]methyl-3-aminopropane-sulfonic
>> >acid)  (Sigma)
>> >60.55g   Tris
>> >3.75g   EDTA
>> >dH2O to 1L
>> >
>> >RUN CONDITIONS
>> >EP Voltage: 4000V
>> >EP Current: 60mAmp
>> >EP Power: 40W
>> >Gel Temp: 42C
>> >Laser Power: 40mW
>> >Run Time: 16 hrs
>> >
>> >We use the long read basecaller, LR-377, from PE (?) Applied Biosystems
>> >3.3.1b2 (available from their web site), with Sequencing Analysis version
>> >3.4.  We aim to have a gel spacing of approx. 14.
>> >
>> >I hope this works as well for you as it has for us.  Let me know if you
>> >have any questions.
>> >
>> >Sincerely,
>> >Glenis
>> >
>> >--
>> >Glenis Wiebe, M.Sc.
>> >University Core DNA & Protein Services
>> >University of Calgary
>> >
>> >tel: (403) 220-4503, fax: (403) 283-4907
>> >e-mail: gwiebe at ucalgary.ca
>> >http://www.ucalgary.ca/~dnalab
>> >
>> >
>> >
>> >Kev wrote:
>> >
>> >> Can anyone send me the recipe for TTE gels for the ABI377 sequencer.
>> >> It's supposed to be in a user bulletin but I haven't received that one.
>> >>
>> >> Any good or bad experiences with them?
>> >>
>> >> Thanks
>> >>
>> >> Kev
>> >>
>> >> ---
>>
>> _/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/
>> Biopolymer Facility
>> Roswell Park Cancer Institute
>> Buffalo, NY   14263
>> Phone: (716) 845-8032
>> Fax: (716) 845-7621
>> Email: biopolymer at sc3101.med.buffalo.edu
>> _/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/
>>
>> ---
>
>
>---
>
>
>

________________________________________________________________

 George Grills
 Director
 DNA Sequencing, Genotyping, 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 at aecom.yu.edu
 DNA Sequencing: http://leper1.ca.aecom.yu.edu/dnacore
 Oligonucleotide: http://sequence.aecom.yu.edu/oligo
________________________________________________________________



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