Message-Id: <2.2.32.19970108175736.006c1d28@mailserver.aecom.yu.edu>
Date: Wed, 08 Jan 1997 12:57:36 -0500
From: George Grills <grills@aecom.yu.edu>
Subject: DNA Seq. Long Ranger
To: Recipients of ABRF List <abrf@aecom.yu.edu>
At 08:19 PM 1/7/97, you wrote:
>I am making my second attempt at using LongRanger at the 4X speed on the
>377. I would like to know the recipe that is being used. LR, TBE, urea,
>water, Temed and APS. Are you degassing and if so for how long. My first
>attempt using the FMC recipe was poor beyong 450 bases compared to my
>cheap, cheap 29:1 acrylamide.
>
>Hal
>
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Hi Hal,
Our Long Ranger gel protocol is listed below. Using this protocol with a 2X
run, our control pGEM template usually gives 750-850 bases sequenced with at
least 98% accuracy. Using this protocol with a 4X run, our control pGEM
template typically gives 600-700 bases sequenced with at least 98% accuracy.
- George
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5% Long Ranger gel (377 Sequencer - 36 cm plates)
Protocol #1 (stock solution):
- 18 ug Urea (Amresco Cat. No. 0378)
- 5 ml of Long Ranger 50% gel stock solution (FMC
BioProducts Cat. No. 50612)
- 5 ml 10X TBE (pH 8.3)
- bring up to 50 ml with dH2O (~ 26 ml)
stir for 1 minute to mix
filter with 2 um Nalgene unit (Nalgene Cat.
No.: 125-0020)
de-gas for 2 minutes
- add 250 ul of 10% Ammonium Persulfate (Amresco Cat. No.
0486), stir gently
- add 25 ul TEMED (Amresco Cat. No. 0761), stir gently
- pour
- polymerize for 2+ hours
Protocol #2 (gel pack):
- use Long Ranger Singel Pack (FMC BioProducts Cat. No. 50691)
- make sure Long Ranger Singel Pack is at room temperature
- remove BLACK clip (=> mix Long Ranger gel solution with
TBE and urea)
- mix contents of compartments well by hand for 1 minute
- place pack on orbital shaker for 5 minutes at medium speed
- mix contents of compartments well by hand again for 1 minute
- place pack on orbital shaker again for 5 minutes at medium
speed
(do the following steps without delays to avoid
polymerization in the pack)
- remove RED clip (=> mix in TEMED)
- mix contents of compartments well by hand for 1 minute
- remove WHITE clip (=> mix in powdered APS)
- drain contents into filter end (=> expose filter to
contents of pack)
- fold pack in half at the line
- tip pack up and cut corner in area marked CUT
- pour solution into beaker (invert and gently squeeze pack
for optimal flow)
- use syringe to pour solution between plates
- polymerize for 2+ hours
___________________________________________________________
George Grills
Director
DNA Sequencing Facility
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@aecom.yu.edu
___________________________________________________________