Here is our protocol for precipitation
We do the sequencing reaction in 5ul since two weeks, before it was in
10ul
We use a multimek to transfere reagents
we premix 500 ml of absolute Ethanol with 20 ml of NaAc pH 5.2 (we add
also a drop about 500 ul of blu dexran to distinguish bottles and to see
the pellet, but it is not necessary and if you decide to add it, do it
before adding NaAc)
than with the multimek we transfere 14 ul of the mix to each well
containing the sequencing reaction.
leave the plate to shake for 15 min then
put it in the centrifuge at 4000 RMP (2000 g) for 35 min.
discard the supernatant by inverting and shaking the plate to eliminate
most of the liquid
dry the plate by leaning it against a piece of paper
wash with 20 ul ehtanol 70%
centrifuge 5 min at max speed
reapeat the wash step
spin the plate in inverted position at minimum g (we use 250 RPM) for
some seconds with a piece of paper below it to eliminate ethanol
residual.
For a 10 ul reation simply duplicate volumes
The result for us is very good
The only drowback is that it takes about
Barbara
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Barbara Simionati
CRIBI Biotechnology Centre
and Department of Biology
University of Padua, Italy
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