TTE gels

Biopolymer Facility biopolymer at sc3101.med.buffalo.edu
Mon Jul 24 14:52:40 EST 2000


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
>>
>> ---





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Biopolymer Facility
Roswell Park Cancer Institute
Buffalo, NY   14263
Phone: (716) 845-8032
Fax: (716) 845-7621
Email: biopolymer at sc3101.med.buffalo.edu
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