Ethanol Precipitation of DNA Cycle Sequencing Reactions
"an old procedure revisited"
version dated 1-24-01
This clearly is an "old procedure revisited" but is extremely
efficient because 1) the amount of resulting nested fragment set
is at least 5 to 10 times more than obtained by Sephadex G-50
filtration and 2) the signal strength
on the ABI 3700 is increased approximately 5 to 10 fold.**
- Take the 384 plate from the thermocycler and add 16ul ddH2O
(depending on reaction volume) to each well using the Hydra.
- Centrifuge to 1000 rpm to force water to the bottom of the well.
Briefly vortex the plate with a tight hold to mix sample with the
water. Centrifuge again.
- Transfer the sample/water mix to a 96 well reaction plate using the
Hydra. Add 60ul EtOH/NaOAc (made according to standard protocol)
to the 96 well reaction plate using a 12 channel pipet.
- Place the 96 well plate in a thermocycle plate base and centrifuge
immediately at 3200 rpm, 4degC, for 30 to 40 minutes. 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.
- Decant the EtOH/NaOAc. This can be done a couple of ways. One way is
turning the plate upside down on a paper towel and patting dry.
However, because we all have a tendency to do this with varing
degrees of force,
a better method is to place upside down on a paper towel in a Beckman CS-6R
table top microtiter plate centrifuge and begin to centrifuge.
immediately turn the centrifuge off. Once stopped, remove the microtiter
plates and place upright on the lab bench.
The important part is to get rid of a majority of the EtOH/NaOAc
and not lose any sample.
- Rince by adding 70ul of 70% EtOH using a 12 channel pipet and centrifuge at
3200 rpm, 4degC, for 10-15 minutes.
- Decant the EtOH
by placing the plate upside down on a paper towel and
centrifuge up to 300 rpm as above and dry in vacuum for 10-15 minutes.
The 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 in a vacuum for 10-15 minutes.
- Dissolve the dryed reaction products in 20 ul of water and load onto
the ABI 3700 as recommended. Sit back and enjoy 5 to 10 times
higher signal strength.
** Note:
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, broe@ou.edu