Ethanol Precipitation of DNA Cycle Sequencing Reactions in 384-well plates
version dated 12-11-2002
This clearly is an "old procedure revisited" but is extremely
1) the amount of resulting nested fragment set
is at least 5 to 10 times more than obtained by Sephadex G-50
2) the signal strength
on the ABI 3700 is increased approximately 5 to 10 fold.**
Note that this is a room temperature ethanol pptn step with
centrifugation at 4degC.
Identical results have been obtained with 1:12 diluted big dye
reactions followed by precipitation at -20degC. In either case, no
'big-dye blob' at ~140 nucleotides was observed, because of
the greatly reduced amounts of big dye in the reaction mixes.
- Take the 384 well viper plate from the thermocycler.
Remove the Silicone Sealing Mat (Axygen Scientific: AM-384-PCR-RD)
and place face down on a paper towel and pat to absorb any liquid
that may be on it. Add 30 ul 95% EtOH/0.12 M NaOAc to the viper
plates using the V-prep protocol with mixing.
- Seal with a Silicone Sealing Mat. Stacking no higher than
four plates, centrifuge at 3200 rpm, 4 degC, for 30 min in the
Beckman CS-6R tabletop centrifuge OR 2400 rpm, 4 degC, for 30
min on the Jouan KR 422 centrifuge.
- Remove the Silicone Sealing Mat and place face down on a paper towel
and as in Step 1. To decant liquid, invert each plate on one paper towel
folded three times. Stack the plates no more than four high (with a paper
towel in between each one and on the bottom) and centrifuge in the Beckman
CS-6R tabletop centrifuge up to 400 rpm and immediately stop.
Once stopped, remove the microtiter plates and place upright on the lab
bench and Discard the paper towels.
The important part is to remove a majority of the EtOH/NaOAc
and not lose any sample.
- Rinse the samples by adding 30 ul 70% EtOH to each well of the
384 well plate using the V-prep protocol without mixing. Seal with
a Silicone Sealing Mat. Then stack the plates in the swinging bucket
of either the Beckman CS-6R tabletop centrifuge or the Jouan KR 422
centrifuge. Stack no more than four plates in each swinging bucket.
Centrifuge at 3200 rpm, 4 degC, for 10 min in the Beckman CS-6R
tabletop centrifuge OR 2400 rpm, 4 degC, for 10 min on the
Jouan KR 422 centrifuge.
- Decant the liquid using the quick centrifugation procedure
described in step 3.
Another 70% ethanol wash can be repeated prior to drying, but only
is necessary if dye blobs are seen during the sequencing run.
- Dry the pelleted reaction products loosely covered with a Kim-wipe
in a vacuum for 20 minutes.
- Cover each plate with a Cyclefoil Storage Plate Sealer
(Robbins Scientific: 1044-39-3) and place the plates in the
gel room freezer to be loaded in the ABI 3700 as recommended.
- Prior to loading the ABI 3700, the dryed reaction products
should be dissolved in 20 ul of water and then loaded onto
the ABI 3700 as recommended. Sit back and enjoy 5 to 10 times
higher signal strength than with the Sephadex cleanup.
It is highly likely that a significant amount of the nested fragment set sequencing reaction
products are bound to the Sephadex G-50 column because of the slight
ionic characteristic of Sephadex when both the column equilibration
and DNA elution are done in the presence of water.
Bruce Roe, firstname.lastname@example.org