Magnetic Bead Cleanup of BigDye Terminator Reactions
MICHAEL L SULLIVAN
mlsulliv at facstaff.wisc.edu
Thu Apr 22 08:42:01 EST 2004
> Has anybody had experience of using the CleanSEQ magnetic beads from
> Agencourt to clean up their BigDye terminator sequence reactions
> beforeloading onto a capillary sequencer (ABI 3700)?
> How does it compare to Gel filtration columns?
> Any comments gratefully appreciated.
I have been using Agencourts CleanSEQ product for a few years now, after our sequencing facility reccommended it for clean up of BigDye sequencing reactions prior to running on their ABI capillary sequencors. I've been pretty pleased with the product. We pretty routinely get back >800 bases of good quality sequence.
One thing to note, though. The Agencourt protocol calls for letting the beads dry following the last wash and prior to resuspension. This actually has a negative impact on sequence quality (big dye blobs at about 100 bp), and our sequencing facility has advised us to go directly to resuspension in water following the wash. I believe Agencourt confirmed to our facility that indeed this is the way it should be done, but I haven't seen that they've made that change in the protocol.
The product seems pricey, but I don't think it really is in the long run. Their starter kit has the magnetic plate and 8 ml of resin. That's enough resin for 800 reactions, and it doesn't seem to go bad (mine's still working well after 3 years in the fridge!) Not counting the magnetic plate, the cost per reaction is about $0.50, I think. I think this is probably less expensive than the Autoseq columns (from Pharmacia) I used to use, but probably more expensive than home made columns. Paying a few pennies more for clean up though saves money if you don't have to repeat reactions or run fewer reactions because of longer reads.
Hope you find this helpful.
PS I have no affilliation with any of the companies named above.
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