EROSENTHAL@BIONET-20.BIO.NET (Eric Rosenthal) (07/07/88)
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 -------
wrp@biochsn.acc.virginia.edu (William R. Pearson) (07/08/88)
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 PearsonGORDON.BRADSHAW@BIONET-20.BIO.NET (Toby Bradshaw) (07/08/88)
Eric: The blur you see at 300-400 bases is due to an anion formed by the glycerol in the Sequenase enzyme when electrophoresed in a buffer containing borate ions. This anion migrates at that position in the gel, screwing up the resolution. I don't normally care about it, but if you do, I would suggest trying a buffer other than Tris-borate or (probably too much trouble) a spin column or precipitation to eliminate the glycerol. As for the wedge gels, a longer fixing time will help since its the failure to remove the hygroscopic urea that makes gels stick to film. Hope this helps. Toby Bradshaw -------
wallace%fmi.ch@RELAY.CS.NET (Andrew Wallace) (07/29/88)
Eric, Regarding your query about the sticking of the X-Ray film to gels, how do you fix your gels before drying ? I get excellent results by using 12%(v/v) methanol/10% (v/v) acetic acid /water, which I apply to the gel by use of a wash-bottle (not leaving the gel swimming in a bath of the stuff - this doesn't work and you risk breaking the gel). The gel and plate are supported above a tray or sink to catch the run-off from the gel, and I just rinse the gel with the fixing solution every 5 minutes for a half- hour. The gel is supported so that it does not sit in contact with old solution. I find that this method gives the best results to remove urea from the gel prior to drying, otherwise the urea crystallises on the surface of the gel, causing the sticking and the little black spots. Best wishes, Andrew Wallace, FMI, Basel, Switzerland. -------