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?
>>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)
>>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
>dH2O to 1L
>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>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?
Roswell Park Cancer Institute
Buffalo, NY 14263
Phone: (716) 845-8032
Fax: (716) 845-7621
Email: biopolymer at sc3101.med.buffalo.edu