Path: utzoo!utgpu!water!watmath!uunet!ig!ames!umd5!uvaarpa!hudson!biochsn!wrp
From: wrp@biochsn.acc.virginia.edu (William R. Pearson)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Uniform gels for reading long sequences
Message-ID: <452@hudson.acc.virginia.edu>
Date: 8 Jul 88 13:41:37 GMT
References: <12412284971.17.EROSENTHAL@BIONET-20.ARPA>
Sender: news@hudson.acc.virginia.edu
Reply-To: wrp@biochsn.acc.Virginia.EDU (William R. Pearson)
Organization: University of Virginia, Charlottesville
Lines: 36

In article <12412284971.17.EROSENTHAL@BIONET-20.ARPA> EROSENTHAL@BIONET-20.BIO.NET (Eric Rosenthal) writes:
]
]	We have been sequencing, using sequenase, and running the reactions
]on a 8% gradient gel.  This gives us 250 to 300 bases worth of sequence.
]In many cases we have then run a 6% uniform gel to try and extend the
]amount of sequence information from the same set of reactions, but we
]have had very mixed results with this.  The gels are often quite blurry
]and we get very little additional sequence information from them.  Does
]anyone have any suggestions for how to improve the quality of these
]extended gels.
]	A related question-- We tryed wedge spacers with these long gels
]and the speration of bands was quite good, but we had terrible problems
]with handling the gels.  In particular, the gels never dryed completely
]on the gel dryer.  Even when we thought they were dry they would stick to
]the X-Ray film and the autoradiographs were often covered with little spots.
]A number of other people have told me that they have had the same problems.
]Does anyone have any tips?
]	Thanks.
]
]						Eric Rosenthal
]-------
	I would try two things:

	(1) Try fixing your gels for 2 - 3X as long (at least 40 min,
possibly 60), making certain that the gel has completely detached from
the lower plate.

	(2) Make certain that you change your buffers every 2 hrs or
so, this will prevent compression at the top of the gel.

We have had better luck with buffer gradient gels than wedge spacers,
but have settled on doing multiple loadings.  We have been running
6% gels for about 6 years.  The blurry problem seems to come and go,
sometimes increasing the amount of fixing time helps.

Bill Pearson