TTE gels

[Glenis Wiebe]

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
>
> ---