Thanks to all who sent in helpful responses to my query regarding the ABI 5x sequencing buffer. It appears that most people use the following recipe. Indeed it seems that it is just 400mM Tris, and 10mM MgCl2, pH 9.0 solution, making it one of the most expensive saltwater solutions in the world. Some of the more helpful responses are pasted below. If you're buying this from ABI, you should complain to the bitterly about their exploitive business practices. Cheers, Paul From: Tomas Hrbek Cycle sequencing 5x replacement buffer (1 liter): 400 ml 1M Tris-HCl pH 9.0 10 ml 1M MgCl2 590 ml milliQ H20 Stir for minimum of 1 hour. Filter though a Watson filter paper. Aliquot into 15ml conical tubes. Store at 4C or room temperature. From: Brant Faircloth the solution that i have for 5X BD dilution Buffer is 400mM Tris-HCl (pH 9.0) and 10 mM MgCl2, just as you wrote. It can be used with BD 3.1, and we run 12th or 16th reactions. S. Lin and Bruce Roe list the same solution for BD 3.1 here (for a 1:64 dilution; 96-well plate premix): http://www.genome.ou.edu/PredispensedDilutedSeqMixBD3orET.html and you can also find the same buffer solution recommended on the (very pretty) barcodeoflife.org site (protocol from the CCDB): http://www.barcodeoflife.org/CCDB_DOCS/CCDB_Sequencing.pdf U. of Washington, however, lists their BD buffer (for 1.1 and 3.1) here: http://dnaseq.bchem.washington.edu/bdsf/help/dilutions.html as (I have shown 5X below): 350 mM Tris (pH 9.0) and 2.5 mM MgCl2 not sure about this one, but I figured you might want to see some options ; ). cheers, b -- Dr. Paul H. Barber Boston University Boston University Marine Program 5 Cummington St. Boston, MA 02215 617-358-4589 office 617-358-4590 lab 617-353-6340 FAX pbarber@bu.edu http://people.bu.edu/pbarber/ Paul Barber