Sequencing Reaction Cleanup in 384 Well Sequencing Reaction Plates

1.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 μl 95% EtOH/0.12 M NaOAc to the viper plates using the V-prep protocol with mixing.

2.Seal with a Silicone Sealing Mat. Stacking no higher than four plates, centrifuge at 3200 rpm, 4 ℃, for 30 min in the Beckman CS-6R tabletop centrifuge OR 2400 rpm, 4 ℃, for 30 min on the Jouan KR 422 centrifuge.

3.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.

4.Rinse the samples by adding 30 μl 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 ℃, for 10 min in the Beckman CS-6R tabletop centrifuge OR 2400 rpm, 4 ℃, for 10 min on the Jouan KR 422 centrifuge.

5.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.

6.Dry the pelleted reaction products loosely covered with a Kim-wipe in a vacuum for 20 minutes.

7.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.

8.Prior to loading the ABI 3700, the dryed reaction products should be dissolved in 20 μl 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.

** 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.

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.**

Note that this is a room temperature ethanol pptn step with centrifugation at 4℃.

Identical results have been obtained with 1:12 diluted big dye reactions followed by precipitation at -20℃. 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.