Molecular Biology Protocols

III. Diatomaceous Earth-based *and* non-Diatomaceous Earth-based Mini-prep

Note: This is a typical mini-prep until step 9, where in step 9a you would precipitate the template and uses it for Taq Cycle Sequencing with the Dye-Labeled Primers, or in step 9b proceed with the diatomaceous earth purification for Taq Dye-Labeled Terminator Cycle Sequencing Reactions.

For Sequenase Dye-Labeled Terminator Sequencing Reactions, use the Midi-prep procedure detailed above.

A. Materials:

[The Prep-A-Gene vacuum manifold, membranes, and related supplies were purchased from BioRad, Inc. A Gast pump was used to generate the vacuum needed for filtration. The pump was protected from aspirated liquid by placing a liquid trap (a 1liter flask with stopper) in line between the pump and the manifold.]

B. Methods:

  1. Place 6 ml aliquot of TB (Terrific broth) media containing the appropriate antibiotic (typically Amp at 25 ug/ml for pUC-based plasmids) into 15ml sterile culture tubes. Using a sterile toothpick or loop, pick individual colonies of the plasmid containing cells into the media containing tubes. Grow these in the shaker (250 rpm) for 16-18 hours at 37oC.

  2. Shake the tubes well and spin the cells down in the culture tubes using the GPR tabletop centrifuge for 5-10 mins at 2500 rpm. After pouring off the supernatant, (some freeze the pellet at -70c for at least 0.5 hour and believe this helps the later cell lysis step).

  3. Resuspend each cell pellet in 200ul of TE containing RNaseA and RNase T1 (50 mM Tris containing 10 mM EDTA, 100 ug/ml RNase A and 50 units/ml RNase T1) *or* resuspend each cell pellet in 200ul of sterile GET/Lysozyme by lightly shaking. If you resuspended in GET/Lysozyme without RNase, be sure to include the RNase step below.

  4. Add 200ul of freshly prepared Alkaline Lysis (NaOH-SDS) solution. Mix gently. Incubate at Room Temperature for 45 min. (some eliminate or reduce this incubation and the yields are similar).

  5. Add 200ul (some prefer 300ul) of 3.0 M sodium acetate, pH 4.8. Mix gently.

  6. Transfer the mixture to 1.5ml microcentrifuge tubes. Then, place the tubes at 4oC (ice bath) or -20oC (in the freezer) for 15min. (Some believe the -20oC incubation improves the quality of the plasmid prep.)

  7. Centrifuge for 15mins at in the cold room, eppendorf-type centrifuge to pellet the cellular debris, protein, precipitated SDS and chromosomal DNA to yield the "cleared lysate".

  8. Transfer the supernatant to a fresh 1.5 ml tube and either incubate again in freezer for 15 min. or centrifuge without incubation as before in the cold room for 15 min.

  9. (a) FOR DYE LABEL PRIMER TAQ POLYMERASE CYCLE SEQUENCING REACTIONS:

    Transfer the supernatant to a fresh 1.5 ml tube and add 1 ml 95% ethanol. Mix by inversion and incubate on ice for 15 min. (some prefer to incubate in the freezer at -20oC for at least 30 min.) Collect the DNA pellet by centrifugation, wash with 70% ethanol, and dry the pellet in the Speed Vac. Dissolve the pellet in 100 or 200 ul or dd-water or 1xTE (10:0.1), * (at present there is a debate going on in my lab regarding dd-water vs * 1xTE (10:0.1), and most have settled on dissolving the ds template in * 200ul of dd-water rather than in TE (10:0.1). In either case the DNA is run on an agarose gel to check the concentration and purity. Use 2 ul for each A, C reaction and 4 ul for each G, T reaction. With double stranded templates, the 30 cycle sequencing program (program 11) is recommended.

    (b) FOR DYE LABEL TERMINATOR TAQ POLYMERASE CYCLE SEQUENCING REACTIONS: To the supernatant add 1ul of RNase A ( 20 ug /ul ) and 1ul of RNase T1 ( 10 units /ul), mix well and incubate at 37oC for 20 mins., and then proceed with step 10).

  10. Following RNase treatment add 1ml of DNA binding matrix (20 mg/ml) to each tube and allow the DNA to bind for 5 mins.

  11. Meanwhile soak the Prep-A-Gene vacuum manifold membrane in isopropanol for at least 3mins. Assemble the manifold apparatus as described in the BioRad Manual.

  12. Turn on the vacuum pump and adjust the vacuum level to 8 inches Hg. Let the membrane dry for 1min and then release the vacuum at the stopcock.

  13. Apply the samples to the wells of the manifold and filter through at 8 inches Hg until all the liquid is filtered through.

  14. With a repeating pipet wash the samples with 250 ul Wash buffer . Repeat the wash three more times allowing all the fluid to filter through between washes.

  15. Reduce the vacuum to 5 inches Hg before turning the vacuum off at the stopcock. Without unscrewing the black clamps release the white clamps and place the collection rack with the clean screwcapped tubes into the manifold. Clamp the manifold with the white clamps . Apply 250ul of Elution Buffer heated to 75oC and pull through at 5 inches Hg. Once all the liquid has filtered through, raise the vacuum to 10-12 inches Hg and let the membrane dry for a minute.

  16. Turn the vacuum off at the stopcock and precipitate the DNA in the screwcapped tubes in the collection rack with 2.5 volumes of ethanol- acetate at -20oC for atleast two hours or overnight.

  17. Spin the DNA in he screwcapped tubes at 12000 rpm for 15 mins at 4oC. Wash the pellet with 500ul of 80% ethanol, dry it and resuspend it in 30ul of 1xTE:10:0.1.
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