Double Acetate Cleared Lysis Procedure for Double-stranded DNA Sequencing Template Isolation using the Biomek 2000 and Automated Hydra96

Required for templates to be sequenced on the MegaBACE but also recommended for the 3700, 377 or LICOR sequencers
Latest update 4-26-00

  1. Cell Growth and Collection:

    1. Pick colonies into 96 well blocks containing 1.5 ml TB/salts containing the appropriate antibiotic. Cover the blocks and shake for 18-20 hours at 350 rpm.
    2. Harvest cells by centrifugation at 1800 rpm for 7 minutes. Pour off the supernatant and allow pellets to drain inverted.

  2. Isolation Protocol:

      Note: The Double Acetate protocol is identical to the Single Acetate isolation for steps 1-13

    1. Suspend the pellets in 200 ul TE-RNaseA/T1 using the Biomek programmed to mix by pipetting the cell solution up and down 20 times. Use the Biomek program, td_disiolv.2 and TE-RNaseA/T1 is: 10 mM Tris-HCl, pH 7.6; 1 mM EDTA; 42 ug/ul RNase A; 0.04 U/ul RNase T1
    2. Add 200 ul of lysis solution to the suspended cells, using "Adding Lysis buffer to..." program on the automated hydra.
    3. Incubate at 37 deg. C in the Incubator Shaker at 250 RPM for 10-15 minutes.
    4. Add 200 ul 3M KOAc or NaOAc, pH 4.5 to each well using " Adding KOAc to ..." program on the automated Hydra. PLEASE RUN THE SOLO WASH PROCEDURE TO CLEAN THE HYDRA SYRINGES AFTER THIS STEP IS COMPLETED
    5. Seal each block with acetate sealer and vortex vigorously until you notice a "tornado" in each well indicating a complete mixing.
    6. Shake at 37 degrees C at 350 rpm for 20 minutes in the Incubator Shaker.
    7. Freeze in -70 degrees C for overnight until solid. This overnight freezing greatly aids in forming a very tight pellet after the subsequent centrifugation step next.
    8. Remove from freezer and thaw. Clear the lysate by centrifugation at 4250 rpm for 45 mins in the Jouan KR422 refrigerated centrifuge. The pellet should be tightly packed and if not, re-freeze and re-centrifuge to obtain a cleared lysate supernatant.
    9. Use "Acetate Transfer (one block at a time)" program on the automated hydra to remove the upper 400 ul of the cleared lysate supernant to another deep well microtiter plate. BE SURE to allow the Hydra to fully wash the needles after each transfer is completed.
    10. Add 1.0 ml Ethanol using the repeater, mix and let stand at room temperature or in an ice/water bath at 4 degrees C (but no lower) for 15-30 minutes. This room temperature ethanol precipitation step results in only a very faint precipitate, rather than the larger precipitate obtained by -70 degrees C incubation. The additional contaminating materials (probably carbohydrate) that precipitate at -70 degrees C, do not precipitate significantly at room temperature, and thus the resulting DNA pellet is smaller but free from significant contamination.
    11. Centrifuge at 3000 rpm for 30 minutes at 4 degrees C in the Beckman CS-6R table top microtiter plate centrifuge.
    12. Decant the supernatant properly and wash with 1 ml 70% ethanol and centrifuge again for 10 to 15 minutes. Dry after decanting the supernatant.
    13. Resuspend the DNA in 200 ul of 10:1 TE pH8.0 and add 100 ul of 7.5 KOAc. Place the blocks in -20 or -70 degrees C overnight.
    14. Allow the blocks to thaw and then centrifuge for 45 minutes at 4250 rpm in the Jouan KR422 refrigerated centrifuge.
    15. Using the hydra transfer the top 200 ul of supernatant to a fresh deep well microtiter plate.
    16. Precipitate the DNA with 500 ul of 100% ethanol at room temperature for 15 minutes and then centrifuge at 3000 rpm for 10 minutes at 4 degrees C in the Beckman CS-6R table top microtiter plate centrifuge.
    17. After decanting the ethanol, wash the pellet with 500 ul of 70% ethanol two times by using the repeater, mixing and centrifuging at 3000 rpm for 30 minutes at 4 degrees C in the Beckman CS-6R table top microtiter plate centrifuge.
    18. Dry the blocks and resuspend the DNA in 100-150 ul of water depending on the DNA yield.
    19. Although typically 2 or 4 ul ul of this ds DNA sequencing template is used in the 1/12 Big-Dye reaction as described in the Big-Dye Protocol a concentration study (using the equivalent of 0.5, 1.0, 2.0 and 4.0 ul of double-stranded template) is advisable.
    Note:
    • The template prepared by this double acetate procedure is suitable for sequencing reactions which are to be loaded on either the MegaBACE or the 3700.
    • There is a folder in the User Protocol directory called "DNA isolation" that has many folders for different number of blocks. It is extremely important to let the hydra auto wash to the full extent of the program.

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      Bruce Roe, broe@ou.edu