Plant Biology Division Protocols
Biomek 2000 Double Stranded DNA Isolation of DNA Sequencing Templates

Updated May 31, 2002

This is an automated version of the double-stranded template DNA isolation procedure that has evolved in Bruce Roe's laboratory over the past several years. The original Biomek 2000 version was developed, programmed, and tested by Judy Crabtree. This final version contains the Noble Foundation modifications for the M. truncatula EST Project. With this protocol, a single Biomek 2000 can isolate four sets of 96 double-stranded DNA sequencing templates in less than 4 hours.

A previous update recommended freezing the samples at -20° C overnight prior to clearing the lysate (step 9). 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 sequencing instrument.

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 37° C with shaking at 350 rpm.

2. The first step is to prepare duplicate glycerol stocks. Place one block on the Biomek tablet in the configuration indicated in the program "Glycerol Stocks." A block containing cell cultures should be placed at position A4; P250 Biomek tips should be placed at position A2; 96-well u-bottom microtiter plates (polypropylene) at positions A5 and A6; reagent quarter module on the left side of B5; tool rack at position A1 with MP200 tool in the right hand rack. At least 25 ml of 30% glycerol should be placed in B5.

3. Start the program. The Biomek will add 100 µl of 30% glycerol to each well of the u-bottom microtiter plates. The Biomek will then transfer 100 µl to each well of the u-bottom plates. Transfer to the u-bottom plates included a mixing step. When completed, heat seal the u-bottom plates and store at -80° C.

4. Harvest cells by centrifugation at 3000 rpm for 10 minutes. Decant supernatant and autoclave before disposal. Cell pellets should be frozen at -20° C.

5. Place 4 blocks containing cell pellets on the Biomek tablet in the configuration indicated in the program "Plasmid 1-4 block". Blocks containing cell pellets should be placed at positions A5, A6, B5 and B6; P250 Biomek tips should be placed at positions A2, A3, B1 and B2; reagent full modules should be placed at positions A4, B3, and B4; tool rack at position A1with MP200 tool in the right hand rack. TE-RNase A+T1 should be placed in A4, SDS/NaOH should be placed in B3, and 3 M Na/K OAc should be placed in B4.
see a screenshot of the dialog box

6. Start the program. The Biomek will add 250 µl TE-RNase A+T1 to each well of the four blocks in two steps and mix 20 times to resuspend. This will use all four boxes of tips. After addition and mixing (1 hour), the Biomek will pause; request that you make sure the pellets have been adequately resuspended (if necessary, vortex the block briefly to breakup any remaining cell clumps) and request that tips to be changed. At this time, all four tip boxes should be changed and "OK" should be clicked.

7. Next, the Biomek will add 250 µl 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."

8. The Biomek adds 250 µl 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 and will return them to the box. Save this box of tips for supernatant transfer (it will reuse the first row of tip for the first transfer). You then remove the four blocks, cover with plate sealers, vortex briefly and place on the shaker at a setting of "6" for 10 minutes (see Trouble Shooting section at the end).

9. Incubate blocks at -20° C. 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) instrument, the blocks should be frozen for at least 8 hours (i.e. overnight), then briefly thawed for 15 minutes at room temperature with periodic shaking (by hand). Centrifuge for 45 minutes at 3000 rpm to pellet the precipitate.

10. Place the blocks back on the Biomek tablet in the configuration shown for the program "Transfer." Centrifuged blocks are placed in positions A4 and A5; clean blocks are placed at positions B4 and B5; P250 tips are placed at positions A2 and A3.

11. The Biomek will transfer the upper 400 µl to clean blocks starting with A4 to B4 using tips at A2. The second transfer is blocks at A5 to B5 using tips at position A3.

12. 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).

13. Incubate at -20° C for at least 2 hours to overnight, centrifuge 3000 rpm for 30 minutes, and decant. Add 500 µl 70% ethanol, centrifuge an additional 15 minutes at 3000 rpm, and decant. Repeat the 70% ethanol wash and dry under vacuum.

14. Resuspend in 100 µl ddH2O (not 10:0.1 TE as EDTA inhibits Taq Polymerase) and assay 2 µl by agarose gel electrophoresis. Centrifuge 3000 rpm for 15 minutes and transfer to a 96-well u-bottom microtiter plate being careful not to transfer any debris.

15. Typically, 3-4 µl of this dsDNA/ddH2O solution is used in each DNA sequencing reaction on the ABI 377, but less (0.2 - 0.5 µl) for the sequencing reactions to be run on the capillary sequencing instruments.

Troubleshooting the Biomek 96-well Plasmid Isolation Protocol
The following difficulties typically depend on the individual doing the prep as there are three major places that folks make mistakes

First, some folks are too vigorous when removing the ethanol and also throw away some ppt DNA.

Second, after the cleared lysate centrifugation step that is used to pellet the genomic and other "crap", the Biomek is programmed to transfer the upper liquid, to another deep well microtiter plate for ethanol precipitation. Here you want to make sure that none of the pelleted material gets carried over. If the top of the pelleted material is not lower than the bottom of the pipet tip, then either mix further to shear more of the genomic DNA and/or re-centrifuge until the pellet is more compressed. Also it is very important that the NaOAc is thoroughly mixed in. More often then not, when you get crud coming over it is because you did not get adequate mixing. Do NOT use a setting less than "6" on the shaker after the addition of the NaOAc.

Third, drying the ethanol ppt'd DNA too long seems to create a problem when trying to re-dissolve the template DNA. Only 10 minutes in a speed vac is needed to suck off the ethanol, and if the resulting DNA is quite difficult to dissolve (especially if it has contaminating genomic that was carried over during the cleared lysate step) you might try reducing the drying time. So we just leave the microtiter plate with the ppt'd DNA out on the bench covered with a Kim wipe for several hours to let the ethanol evaporate.

Bob Gonzales     
(plagiarized from Bruce Roe)