5X buffer, etc.

[Amy Avery]

1. Does anyone know the recipe for ABI's 5X sequencing buffer?

2. Although we close our rxn tubes tightly (USA Scientific 8-tube strips),
sometimes we have a problem with evaporation. Are there better tubes out
there?

Thanks in advance
Amy




Here are the responds to the question regarding G-Blobs:

From: Molly_A._Rodgers at ccmail.bms.com (Molly A. Rodgers)
Subject: Re: A few questions
To: aavery at bidmc.harvard.edu
Mime-Version: 1.0
Status: RO
X-Status:

Content-Type: text/plain; charset=US-ASCII
Content-Transfer-Encoding: 7bit
Content-Description: cc:Mail note part

     We also use Millipore clean up plates with sephadex but after spinning
     the samples through the columns we also do an isopropanol
     precipitation with 80ul of 75% isopropanol per reaction. This seemed
     to get rid of the extra dye blobs.

     As for the 700 bases from a 4x run,  have you tried ABI's protocol for
     using 40% 29:1 acrylamide:bis? This extended our reads out to about
     700 using the 3 and a half hour run.

     hope this helps!
     Molly


Hi Amy,
	Three quick comments that you might consider:
1. save those FS mixes.  We and others have noticed that some regions
   that we used to be able to sequence through with the dRhodamines
   cannot be read through with the Big-Dyes.  It's not that many but
   enough to make it worth your while to save the dRhodamine kits away
   for a "rainy day".
2. I know it sounds stupid and I know you've got more G-50 in those
   wells of the filter plate that should be more than sufficient to
   remove the dyes, but we've seen the same thing if you do not really
   "top off", i.e. fill those wells to the brim, with G-50.  Check
   out our protocol at URL:
	http://www.genome.ou.edu/proto.html
3. There is so much variation between other peoples samples that I would
   suggest you do the experiment with your templates/reactions regarding
   your question about 2X vs 4X, etc.  It might be useful to see this
   data yourself to make a decision rather than relying on others in this
   case.
Cheers........bruce
*************************************************************************
Bruce A. Roe, Ph.D  George Lynn Cross Research Professor
                    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/ ************************