This is an automated version of the double-stranded template DNA isolation procedure that has evolved in my laboratory over the past several years. This Biomek 2000 version was developed, programmed, and tested by Judy Crabtree. Thanks go to Adonis Reece, Guozhang Zhang and Steve Toth for their suggested modifications during the early testing phase. With this protocol, a single Biomek 2000 can isolate four sets of 96 double-stranded DNA sequencing templates in less than 4 hours.
The September 26, 1999 update recommends freezing the samples at -20 deg C overnight prior to clearing the lysate (step 7). This longer freezing results in tighter pellets and less contamination of the supernatant with insoluble material that has a tendency to clog the capillaries on the new ABI3700 and MegaBACE sequencing instruments
1. Pick colony harboring plasmid of interest into 96 well square well block containing 1.5 ml TB+Salts with appropriate antibiotic. Grow 22-24 hours at 37degC with shaking at 350 rpm.
2. Harvest cells by centrifugation at 2500 rpm for 7 minutes. Decant supernatant and autoclave before disposal. Cell pellets may be frozen at -20degC if necessary.
3. Place 4 blocks containing cell pellets on the biomek tablet in the configuration indicated in the program dsisol.v1. Blocks containing cell pellets should be placed at positions A5, A6, B5 and B6; P250 biomek tips should be placed at positions A2, A3, B2 and B3; reagent half modules should be placed at positions A4 and B4; tool rack at position A1with MP200 tool in the right hand rack; position B1 is empty. TE-RNase A+T1 should be placed in the left side of A4, SDS/NaOH should be placed in the right side of A4, and 3 M Na/K OAc should be placed in the left side of B4. The right module of B4 is empty.
4. Start the program. The biomek will add 200 ul TE-RNase A+T1 to each well of the four blocks and mix 20 times to resuspend. This will use all four boxes of tips. After addition and mixing (1 hour, 6 minutes), the biomek will pause and request tips to be changed at position A2. At this time, all four tip boxes should be changed and "Replace All" should be clicked.
5. Next, the biomek will add 200 ul SDS/NaOH and will mix an additional 10 times. This will use all four boxes of tips. The biomek will pause and request tips to be changed at position A2. Change tips at A2 only and click "OK."
6. The biomek adds 200 ul 3M NaOAc, pH 4.8 (or 3M KOAc, pH 4.8) to the blocks. In doing this, the biomek only uses one row of tips. Leave this box of tips on the biomek...it will reuse the row of tips for the first supernatant transfer. You then remove the four blocks, cover with plate sealers, vortex briefly and place in 350 rpm shaker for 10 minutes.
7. Incubate blocks in -20degC for 15-30 minutes and centrifuge for 45 minutes at 3000 rpm to pellet the precipitate. We recently have observed that a longer freezing step results in a more well defined pellet. Thus, especially for DNA sequencing templates being used on the capillary (ABI 3700 or MegaBACE 1000) instruments, the blocks should be frozed for at least 8 hours (i.e. overnight), briefly thawed for 15 minutes at room temperature with periodic shaking (by hand) and then centrifuged as above.
8. Place the blocks back on the biomek tablet in the new configuration as shown on the screen. Centrifuged blocks are placed in positions A5, A6, B5 and B6; clean blocks are placed at positions A3, A4, B3 and B4; P250 tips are placed at positions A2, B1 and B2.
(NOTE: The graphical display of the new configuration does not show tips at B1, but transfer requires tips at this position. This is just a programming glitch).
9. The biomek will transfer the upper 400 ul to clean blocks starting with A5 to A3 using tips at A2. The second transfer is blocks at A6 to A4 using tips at position B1. Then the biomek will ask for new tips to be replaced at position A2. Replace the tips at A2 and click "OK." The third transfer is blocks at B5 to B3 using tips at A2 and the fourth transfer is B6 to B4 using tips at position B2.
10. Ethanol precipitate by adding 1 ml 95% ethanol to each well of the block and cover with plate sealer (do not invert as the ethanol will degrade the glue of the sealer).
11. Incubate at -20degC for 30 minutes to overnight, centrifuge 3000 rpm for 30 minutes, decant. Add 500 ul 70% ethanol, centrifuge an additional 15 minutes at 3000 rpm, decant and dry under vacuum.
12. Resuspend in 100 ul ddH2O (not 10:0.1 TE as EDTA inhibites Taq Polymerase) and assay 2 ul by agarose gel electrophoresis.
13. Typically, 1-2 ul of this dsDNA/ddH2O solution is used in each DNA sequencing reaction on the ABI 377, but less (0.2 - 0.5 ul) for the sequencing reactions to be run on the capillary sequencing instruments.
This isolation of 4 blocks requires 100 ml of each reagent (TE-RNase A+T1, SDS/NaOH, 3M NaOAc, pH 4.8).
Bruce Roe, firstname.lastname@example.org