G. 96 well double-stranded template isolation

A manual as well as an automated procedure is given below. The automated method is a modification of a previously reported procedure (4) which allows simultaneous isolation of 96 double stranded DNAs per Biomek 1000 Automated Laboratory Workstation within two hours. Basically colonies containing double-stranded plasmids are picked with sterile toothpicks into media and incubated at 37degC for 24 hours with shaking at 350 rpm. These cells are harvested by centrifugation and the pellets are either manually or robotically resuspended by the addition of TE-RNase solution. An alkaline lysis solution is used to lyse the cells and the lysate is precipitated with KOAc. The lysate is cleared by filtration and further concentrated by ethanol precipitation. An aliquot from each DNA sample is subjected to agarose gel eletrophoresis to crudely assay concentration and purity. The yield of double stranded template is approximately 3 mg per sample.

Protocol

Manual Double stranded isolation method

The following is a manual, 96 well, double stranded sequencing template isolation procedure that has been developed in our laboratory. A similar procedure that has been automated on the Biomek is presented elsewhere herein.

1. Pick individual shotgun clones off a plate with a steril tooth pick and deposit each separately into 96 well block containing 1.75 ml of TB media per well. Keep toothpick in media for about 5 minutes to allow the cells to defuse into the media, remove the toothpicks, cover the 96 well block with the loose fitting lid, and allow the cells to grow for 24 hours in the 37degC shaker/incubator at 350 rpm.

2. Remove block from the shaker/incubator and collect the cells by centrifugation at 2500 rpm for 7 minutes. The cells can be stored frozen at -20degC in the block at this stage.

3. After thawing the cells, add 100ul TE-RNAse-A solution containing RNAse T1, mix by pipetting up and down 4-5 times to resuspend the cell pellet and then incubate in the 37degC incubator/shaker for 5 minutes at 350 rpm to mix more thoroughly.

4. Remove the block from the incubator/shaker and then add 100ul of alkaline lysis solution. Shake the block by hand to mix the reagents and then incubate at room temperature for 1 hour with intermittent swirling. 5. Then add 100ul of either 3M potassium or sodium acetate, pH 5, and place the block in the 37degC shaker/incubator for 5 minutes at 350 rpm to thoroughly mix and shear genomic DNA to reduce the viscosity of the solution. Place the block at -20degC for 30 minutes.

6. Centrifuge the block in the GPR centrifuge at 3000 rpm at 4degC for 30 minutes.

7. Carefully remove 200 ul of the supernatant from each well in the 96 well block with the 12 channel pipetter and transfer them to a v-bottom microtiter plate, being careful not to transfer any cell debris.

8. Transfer 10 ul of supernatant into the respective cycle sequencing reaction tubes, and precipitate with 150 ul of 95% ethanol (without added acetate). After storage at -20degC for 30 minutes, the pellet is collected by centrifugation, washed three times with 70% ethanol, and dried directly in the cycle sequencing reaction tubes.

9. Prior to adding the fluorscent terminator cycle sequencing reaction mix, the dried templates should be stored at -20degC. An additional 75 ul of the supernatant is transfered to a Robbins PCR reaction tube (in 96 well tube format) and precipitated with 200 ul of 95% ethanol, washed three times with 70% ethanol, and stored dry at -20degC for future use.

The following is an automated, 96 well, double stranded sequencing template isolation procedure that has been developed in our laboratory.

1. Pick colonies using a toothpick into 1.8 ml TB with TB salts containing appropriate antibiotic and shake for 22-24 hours at 350 rpm in a 96 well block with cover.

2. Harvest cells by centrifugation at 1800 rpm for 7 min. Pour off supernatant and allow pellets to drain inverted. Cell pellets may be frozen at this point if necessary.

3. Turn on Biomek, begin the program DSISOL2 and set up the Biomek as indicated in the configuration function on the screen. Specifically, you should put TE-RNase solution in the first module, alkaline lysis solution in the second reagent module and 3 M KOAc, pH 4.8 in the third module.

4. Place the 96 well block containing cells onto the Biomek tablet at the position labeled "1.0 ml Minitubes". Place a Millipore filter plate in the position labeled "96well flat bottomed microtitre plate".

5. Press ENTER to continue with the program.

6. First the Biomek will add 100 ml TE-RNase solution to the cell pellets and mix to partially resuspend.

7. Next, the biomek will add 100 ml alkaline lysis solution to the wells of the filter plate.

8. The biomek then will mix the cell suspension again, transfer the entire volume to the filter plate containing alkaline lysis solution, and mix again. Set up the filtration apparatus with a clean 96 well block to collect the filtrate (wash and reuse the block used for growth).

9. The biomek will add 100 ml 3M KOAc, pH 4.8 to the wells of the filter plate and mix at the sides of the wells. Some choose to place the filter plate at -20degC for 5 minutes at this point. Transfer the filter plate to the QiaVac Vacuum Manifold 96 and filter using water vacuum only (do not do a harsh filtration as the plates are fragile and will loose their seal). This will typically take less than 20 minutes.

10. The supernatant collected in the 96 well block is the crude DNA and must be ethanol precipitated before use by the addition of 1 ml 100% ethanol and incubation at -20degC for at least 30 minutes.

11. Centrifuge for 25 minutes at 3000 rpm in a cooled Beckman GPR centrifuge.

12. Decant and wash with 500 ml 80% ethanol and centrifuge for an additional 5 minutes at 3000 rpm.

13. Decant the supernatant, drain inverted on a paper towel. Dry under vacuum.

14. Resuspend in 50 ml 10:0.1 TE for use in dye primer or dye terminator sequencing chemistry.