dsisol2000.txt

Biomek 2000 Double Stranded DNA Isolation of DNA Sequencing Templates

Updated September 26, 1999

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.

Reagents:

This isolation of 4 blocks requires 100 ml of each reagent (TE-RNase A+T1, SDS/NaOH, 3M NaOAc, pH 4.8).

TE-RNase A+T1

50 mM Tris-HCl, pH 7.6
10 mM EDTA, pH 8.0
40 ug/ml RNase A
40 U/ml RNase T1

SDS/NaOH

0.2 N NaOH
1% SDS

3M NaOAc, pH 4.8

408.24 g NaOAc-3H2O
Dissolve in 300 ml ddH2O, adjust pH with glacial acetic acid and bring final volume to 1L with ddH2O. (Does not go fully into solution until addition of 500 ml or so of glacial acetic acid...must stir and pH simultaneously).

Programs present on OU_Biomek2000_PROTOCOLS disk:

dsisol.v1 The full length procedure for isolating double-stranded DNA from 4 blocks of culture.
Because experience has taught us that sometimes machines crash, the full length program is broken into several component parts, described below, such that one may start at the beginning of any step along the way. The tip usage should be identical to that in dsisol.v1.
SDS_to_end: this routine begins at step 5 in the procedure, assumes that the cells are fully resuspended in TE-RNAse A+T1, and continues to the end of the method. Tip usage is identical to that in dsisol.v1.
acetate_to_end: this routine begins at step 6 in the procedure, assumes that the cells have been treated with TE-RNase A+T1 and SDS/NaOH, and continues to the end of the method. Tip usage is identical to that in dsisol.v1.
transfer: this routine begins at step 8 in the procedure and allows for transfer of the supernatant to clean blocks. The initial configuration that shows up in this method looks like the configuration for the beginning of the dsisol.v1. This is okay, but ignore this configuration. The biomek will align and then show you the configuration required for the transfer of supernatant. The tip usage is identical to that in dsisol.v1.

To view the latest (4-1-99) instructions for downloading the Biomek 2000 version of this program, click on the line below.

OU_Biomek2000_PROTOCOLS

Troubleshooting the Biomek 96 well Isolation

When I am training my students to use the Biomek procedure, I always have them do a 96 well block of pUC the first couple of times. That way I kill two birds with one stone. First, they get some good standards and second if they screw up, it's not some valuable sample that's gone down the tubes. Here we grow 250ml of culture in a 1liter flask overnight and then distribute alaquots to each of the 96 wells in the deep well microtiter plate for isoln.

Then, do a concentration curve on the standard pUC to get a feel for how much template is necessary. In fact, I definitely suggest that doing a template DNA concentration curve is mandatory, especially for folks who are new at this procedure. We typically dissolve our isolated template in 50 ul of ddH2O. It seems important to use ddH2O instead of TE as the EDTA inhibits the sequencing reaction.

Our typical concentration study uses 1, 2, 3, 4 ul of template in a 5-7 ul reaction containing 2ul of the TaqFS kit and 1-2 ul of primer (typically 6.5 to 12 pmoles). We've found that using this one-quarter reaction works quite well and saves money, but the final volume can be between 5 ul and 7 ul without any noticable effect.

It also is advisable to repeat the isolation several times, as it does take some getting used to since even though there are not alot of manual steps, folks to things differently even if they are following the same protocol.

Also, please note that the yield in each plate/well may vary as some inserts seem to inhibit cell growth and/or plasmid copy number. Thus, doing a concentration curve on selected samples may help.

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.

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 speedy 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. Some 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.

Bruce Roe, broe@ou.edu