Maureen Dolan writes:
=>
=> We have traced the aberrations in this region of the gel to old or
=> improperly de-ionized formamide used in preparation of the loading dye.
=> We now store the formamide (Amresco) over Biorad's AG501 resin
(1g/100ml)
=> at 4 C, replace this stock bottle every 3 months and make fresh
=> formamide/dye mix for each day's runs. It may be overkill but we
=> haven't seen any loss of signal or resolution in the 150-250 nt
=> region of the gel since adopting this practice.
=>
=>
=> --
=> Maureen Dolan, Ph. D.
=> Department of Molecular Biology
=> Massachusetts General Hospital
=> Wellman Bldg. 1131
=> 50 Blossom Street
=> Boston, MA 02114
=>
=> Office: (617) 726-5930 Lab: (617) 726-5938 FAX: (617) 726-6893
Maureen,
Thanks for the tip. FYI, our recipe for "ABI loading dye" is:
Deionized Formamide:
Stir formamide (Schwarx/Man Biotech 800686) with Amberlite MB resin
10g/100ml for 1 hr, filter through Whatman 3MM, store in a dark bottle
at rm. temp or 4degC.
0.5M EDTA, pH 8.0:
Dissolve 186.1 gm of Na2EDTA in approx. 400 ml of ddH20, adjust to
pH 8.0 with 10 M NaOH, and adjust the final volume to 1 l with ddH20
100mM EDTA, pH 8.0 with Blue Dextran:
20 ml of 0.5 M Na2EDTA, approx. 3 gms Blue Dextran, and 80ml ddH20
to make 100ml.
FE (formamide-EDTA) with Blue Dextran:
Make fresh using 10 ul ddH20, 10ul 100mM EDTA pH 8.0 containing blue
dextran, and 100ul of deionized formamide.
One "interesting" note is that periodically a new student doesn't seem to
realize that there is a difference between the loading dye for the ABI
PAGE gel and an agarose gel (where 10X Agarose gel loading dye is:
1.5g Ficol (Sigma F-2637), 0.02g bromophenol blue (Sigma B-0126), 0.02 g
of xylene cyanole FF (Kodak T1579) brought to 10ml with ddH20 and store
at -20degC.). Try this if you want to see really cool bands on your
ABI gels just for grins.............
Happy sequencing.........bruce
***************************************************************************
Bruce A. Roe, Ph.D Department of Chemistry and Biochemistry
University of Oklahoma, Norman, OK 73019-0370,
U.S.A.
Phone: (405) 325-4912 or 7610; FAX: (405) 325-7762; e-mail: broe at ou.edu
********************** http://www.genome.ou.edu/
**************************
