peak stretching

Bruce Bruce
Mon Feb 10 09:40:40 EST 1997

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 
=> 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

	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, 
Phone: (405) 325-4912 or 7610;  FAX: (405) 325-7762;  e-mail:  broe at ou.edu
********************** http://www.genome.ou.edu/ 

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