DIATOMACEOUS EARTH-BASED PLASMID DNA PREPARATION

We had a number of problems in obtaining significant yields of a 10 KB plasmid from the E. coli strain XL-1 Blue. Bob Horowitz found the following protocol, modified it slightly, and used it successfully. The original protocol was downloaded from Northwest Fisheries Science Center, U.S. Dept. Commerce, Molecular Biology Protocols. Our thanks to them for making this public.

Procedure
  1. Grow 1 liter of bacteria conta ining the desired plasmid, cosmid, or P1 chimeric vectors. Double the volumes below for 2 L of bacteria. Pellet the bacteria in a centrifuge at 4200 xg for 10 min at 4°C in 250-500 ml centrifuge bottles (5000 rpm in Sorvall GS-3 rotor). Resuspend the bacteria in 50-100 ml of solution #1. Transfer to a 250 ml clear plastic centrifuge bottle. Centrifuge again at the same speed and discard the supernatant. The cells may be frozen at this point.

  2. If the cells were froze n, let them thaw at room temperature. Resuspend them in 17.5 ml of GET/LYSOZYME solution using a metal spatula. Do not vortex at any time during the procedure because you will shear the chromosomal as well as the plasmid DNA. The total volume at this point will be close to 20 ml.

  3. Add 35 ml of ALKALINE LYSIS solution. Mix gently with a spatula and incubate on ice for 5 min. The solution becomes very viscous and stringy.

  4. Add 26. 3 ml of 3M sodium acetate, pH 4.8. Mix gently with a spatula and incubate on ice for 20 min. (The original protocol called for 60 min.)

  5. Remove the precipitated SDS, protein and chromosomal DNA by filtering through double layered cheesecloth, gauze or fine mesh nylon. Centrifugation at 12,000xg (17,200 rpm in an SS-34 rotor or 10,300 rpm in a GSA rotor) for 10 min at 4°C. This may be repeated if required (we only did it once). (Centrifuging first and then filter ing the supernatant also works well. You will have about a 70 ml volume at this point.)

  6. To the cleared supernatant add DNase-free RNase A to a final concentration of RNase A of 40 µg/ml. Our stock is 10 mg/ml RNAse A. (The original protocol also called for adding RNase T1 to 40 units/ml). Incubate at 37°C for 30 min.

  7. Add an equal volume (about 70 ml) of ISOPROPANOL to the RNase treated lysate. Incubate at room temperature for 5 min and centrif uge at 12,000xg (Sorvall RC5-B: 10,000 rpm in an SS-34 rotor or 8750 rpm in a GSA rotor) for 15 min, 4°C.

  8. Resuspend the pellet in 5 ml (final volume will be about 6 ml) of TE. Add 2 volumes of 100 mg/ml diatomaceous earth matrix in guanidine HCl buffer. Allow the DNA to bind for 5 min. Centrifuge the DNA-bound matrix at 12,000 xg at 4°C (Sorvall RC5-B: 10,000 rpm in an SS-34 rotor) for 5 minutes.

  9. Decant the supernatant and wash the pellet with 2 volumes (i.e. 2 x 10.6 ml) of WASH BUFFER. Gently resuspend the pellet by inversion. Centrifuge at 12,000 xg at 4°C for 5 minutes.

  10. Decant the supernatant and wash the pellet with 2 volumes (i.e. 2 x 10.6 ml) of ACETONE. Gently resuspend the pellet by inversion. Centrifuge at 12,000 xg in an SS-34 rotor, 4°C for 5 minutes.

  11. Decant the supernatant and dry the pellet in the vacuum oven and/or at 65°C in a water bath until dry.

  12. Elute the DNA from the dried matrix with 1 volume of ELUTION BUFFER (i.e. 1 volume approximately the size of the pellet, 5-8 ml) at 65°C for 10 min with intermittent stirring every 2 - 3 minutes. Do not vortex. Centrifuge at 12,000 xg for 5 min and collect the supernatant.

  13. Repeat step 12 for increased yield of vector.

  14. Precipitate the DNA with 2.5 volumes of ETHANOL ACETATE at -20°C or on dry ice. Centrifuge for 15 min at 12,000 xg (10,000 rpm in an SS-34 rotor) at 4°C. Wash the precipitated DNA with 1 volume of 70% ETHANOL and dry the pellet under vacuum.
  15. Resuspend the DNA pellet in 2ml of ELUTION BUFFER.
   Average yield in original protocol:
      P1 or Cosmid: 0.2 - 2µg per ml culture.
      Plasmid (pUC or related clones): 2 - 4µg per ml culture.
& nbsp;     Our yield was 2 - 6 mg/L on 3 plasmid preparations.

RECIPES

Solution #1 (0.1M NaCl; 50mM Tris-HCl pH 7.8; 10mM EDTA):
20 ml 5 M NaCl
50 ml 1 M Tris-HCl, pH 8.0
20 ml 0.5 M EDTA, pH 8.0
Use HCl to pH to 7.8 (takes approximately 1 ml of concentrated acid). QS to 1 liter with water and autoclave.
GET/LYSOZYME sol ution:
50 mM glucose (0.9 g d-glucose)
10 mM EDTA (1 ml of 1M stock)
25 mM Tris-HCl, pH 8.0 (2.5ml of 1M stock)
QS to 100 ml with water.
Make this solution fresh, or filter sterilize before storing at 4°C. Add 2 mg/ml lysozyme (0.2 gm lysozyme/100 ml GET) just before use.
NaOH/SDS (Alkaline lysis solution) (0.2 N NaOH, 1% SDS):
< !-- Row 4 Column 1 -->
Stock Solution 100 ml 35 ml
1 N NaOH 20 ml 7 ml
20% SDS 5 ml 10 ml
Distilled water 75 ml 18 ml
Make this solution fresh.
3.0 M Sodium Acetate, pH 4.8:
24.61 g sodium acetate
Approximately 50 ml glacial acetic acid
QS to 100 ml with water after the desired pH is reached.
This pH is very important! More than half of the volume is concentrated acetic acid.
Diatomaceous Earth DNA Binding Matrix:
10 g of diatomaceous earth (Sigma D-5384)
Resuspend in 100ml of double distilled water in a 100ml measuring cylinder and let it settle for 3 hours. Decant the supernatant with the fines. The matrix packs fairly tight here, but be careful not to resuspend it. To store the de-fined matrix, resuspend it in TE and transfer it to a screw cap bottle. Store at 4°C . For the DNA extraction, resuspend an aliquot of the de-fined diatomaceous earth in 6M guanidine hydrochloride buffer for immediate use. The final matrix concentration is about 100mg/ml assuming no significant losses during the defining step.
Guanidine HCl Solution: 6 M guanidine-HCl; 50 mM Tris-HCl, ph 8.0; 20 mM EDTA
57.32 g guanidine-HCl
5 ml 1 M Tris-HCl, pH 8.0
4 ml 0.5 M EDTA
Heat slightly to dissolve. QS to 100 ml with water. Autoclave.
Wash Buffer:
10 mM Tris-HCl, pH 8.0 (1 ml of 1M stock)
1 mM EDTA, pH 8.0 (0.1 ml of 1M stock)
50% ethanol (50 ml absolute alcohol)
Q.S. to 100 ml with double distilled, autoclaved water.
Elution Buffer (TE:10:1):
   **Note** This Tris-EDTA ratio is 10:1, not the usual 10:0.1
10 mM Tris-HCl, pH 8 .0 (1 ml of 1M stock)
1 mM EDTA, pH 8.0 (0.1 ml of 1M stock)
Make the volume to 100 ml with double distilled water.
Ethanol Acetate:
95% Ethanol (95 ml absolute ethanol)
0.12 M sodium acetate (4ml of 3M sodium acetate, pH 4.8)
Make the volume to 100ml with double distilled water.
DNase-free RNase A:
(a) For 100ml of RNase A buffer:
0.1 M sodium acetate (0.82 g anhydrous)
0.3 mM EDTA (0.3 ml of 100 mM stock)
Adjust pH to 4.8 with acetic acid. Q.S. to 100ml with double distilled water. Aliquot in 2 ml amounts and store at -20°C. Add 40 mg of pancreatic RNase A to 2ml aliquots of RNAse A buffer just before use and inactivate any contaminating DNase by heating at 80°C for 10 min.
(b) RNase T1 (10 units/µl) made in 50mM Tris-HCl pH 7.6.
    Send comments and updates to  Dr. Bart Frank, Arthritis and Immunology Program, OMRF
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The original protocol on which this was based was downloaded from Northwest Fisheries Science Center, U.S. Dept. Commerce, Molecular Biology Protocols. Our thanks to them for making this public. Proper citation for the slightly modified protocol presented here is: "Frank, M. B. Diatomaceous Earth-Based Plasmid DNA Preparation, Modifications to a Northwest Fisheries Science Center Protocol. In: Frank, M. B. ed. Molecular Biology Protocols. (http://omrf.ouhsc.edu/~frank/diatom.html). Oklahoma City. Revision Date: April 24, 2000."