Dr. Duncan Clark
Duncan at nospam.demon.co.uk
Wed Aug 30 04:23:33 EST 2000
In article <8oh5op$4d2$1 at nnrp1.deja.com>, ashiflet at my-deja.com writes
>Does anyone have any suggestions on optimizing PCR reactions when
>going from a 96-well format to a 384-well format? We are trying to
>amplify 1 - 4 kb genomic inserts from pBluescript which were
>supplied to us in 384-well format. We had some earlier copies in
>96-well plates which we successfully amplified, however we would like to
>amplify from the 384-well plates because they contain more clones. Any
>suggestions would be welcome! We are using an MJ Cycler, PTC-200.
What I would do is to use a Taq:proofreading enzyme mix for the PCR and
use re-designed M13 universal and reverse primers i.e. something
flanking the polylinker region with a Tm of >65C. Then run a 2 step PCR
94C to 65C. Use 1.25u enzyme per 50ul PCR. The mix will be more
efficient for the PCR and more forgiving of sequence that is being
PCR'd. Prepare a Master Mix of all reagents bar the template.
How were the clones provided i.e. liquid E.coli cultures in the 384 well
plate or what? If they are liquid cultures then 1ul would be ample in a
25ul PCR. Don't use too much, for fear of inhibiting the PCR, and run
say 25-30 cycles of PCR. That is overkill in cycles but at this stage
will give you a clear indication of clones with inserts if you run say
5ul of the PCR on a gel. You could always save more time and run the PCR
with a dye loading buffer in it so you can run the PCRs immediately out
on a gel without having to take out aliquots add stopper then load etc.
You could reduce the no. of PCRs by pooling 1ul aliquots from each row
i.e. take 1ul from each well in row A and pool, repeat for Row B etc.,
and each column then run PCRs on 1ul from those individual pools. From
the agarose gel you should be able to work out that if Row A and Column
4 gave the same size PCR product then A4 has an insert etc. If the
majority of your clones have inserts then don't bother - you could get
too complicated a pattern to work it out!
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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