Frazer Murray wrote:
> Hi everyone
> has anyone else been having trouble with their sequencing reactions
> lately? I've found I've had to increase the amount of sequencing reagent
> from 2ul to 3ul (in 10ul total volume) which has considerably increased
> costs. I've started on a new library but the old one was showing the same
> problem so I don't think that was the cause.
> It would be interesting to hear if others had noticed this as well.
> [...]
We use the same volume as you, but 1 ul of Big Dye reaction mix (1/8th rxns).
We pretty much use the protocol from Bruce Roe's web page:
http://www.genome.ou.edu/big_dyes_plasmid.html
But we stay at 1/2 volume (10 ul). Basically the idea there is that you can
reduce the volume of the reaction and/or you can reduce the concentration of
the Big Dye Mix in the reaction. But if you reduce the concentration of the
Big Dye Mix in the reaction, you adjust the buffer and MgCl2 concentration
back to where it should be with the so-called "5x ABI" buffer (400 mM
Tris-HCl, pH9/10 mM MgCl2) *and* to recover from the loss of signal you would
otherwise observe: increase the number of cycles of amplification. We use 99
cycles (11 hours on an AB 9700) because we run the thermal cycling overnight
anyway.
I should note here that I could not get this protocol to work initially. Two
other factors come into play. One is that it does not work well in my hands
unless I also use DMSO (5% final volume). The other is that I think small
amounts of alcohols (or other volatiles?) in the DNA preps have an impact on
the quality of reduced concentration reactions. I write this because I
noticed that using full strength reactions one can sequence minipreps with a
considerable amount of alcohol in them--enough that you can smell the
alcohol. But those same preps don't work for 1/4th reactions--unless I
re-precipitated the DNA. Now we resuspend our DNA pellets in open containers
on a 37 oC shaker. I think this helps because the volatiles will
preferentially evaporate.
Phillip San Miguel
Purdue Genomics Core Facility
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