gwiebe at ucalgary.ca
Wed Jul 26 17:28:14 EST 2000
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.
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.
> >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
> >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
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