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

For templates to be sequenced on the 3700, 377 or LICOR sequencers
Latest update 2-29-00

  1. Cell Growth and Collection:

    1. Grow subclones for 18 to 20 hours with shaking in the New Brunswick Incubator Shaker at 350 RPM.
    2. Centrifuge at 2900 rpm for 10 minutes and freeze at -70 degrees C for at least two hours.

  2. Isolation Protocol:

    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 200 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 0.5ml 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 15 minutes. Dry after decanting the supernatant and suspend the isolated double- stranded DNA sequencing templates in 100 ul sterile distilled-deionized water.
    13. Typically 2 ul of this ds DNA sequencing template is used in the 1/12 Big-Dye reaction as described in the Big-Dye Protocol.

    • The template prepared by this double acetate procedure is suitable for sequencing reactions which are to be loaded on the 3700, but is not of sufficient purity for the MegaBACE.
    • 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,