diatom_earth_prep.txt - Roe lab protocol

[Bruce Roe]

Hi all,
	I've been pretty quiet with all this discussion of Magic Mini preps.
The recent re-posting of the "Merlin procedure" and at the request of several
others, I am posting here our "cookbook" for using diatomaceous earth for maxi,
midi, and mini-preps.  The DNA prepared as described in this protocol (when 
followed by a capable scientist) results in DNA that works extremely well for
sequencing (see comments in the protocol), restriction enzyme digestion and 
transformation.  The DNA is stable when frozen and thawed a number of times and
the Maxi prep. has been successfully used to isolate chimeric plasmids, cosmids
and p1 constructs in the size range from 2.4kb to 100kb.

        We have not used this protocol for isolating ss m13 templates as
we previously published a robotic method for isolating 96 m13 templates in
less than 3 hours and here are the references for the robotic m13 isolation
procedure.

E.R. Mardis and B.A. Roe. Automated Methods for Single-Stranded DNA Isolation
and Dideoxynucleotide DNA Sequencing Reactions on a Robotic Workstation.
BioTechniques 7, 840-850 (1989).

S. L. Chissoe, Y.F. Wang, S.W.  Clifton, N. Ma, H.J. Sun, J. S. Lobsinger, S.
M. Kenton, J. D. White and B. A. Roe. Strategies for Rapid and Accurate DNA
Sequencing. Methods: A Companion to Methods In Enzymology. 3, 55-65 (1991).

        So here it is.  Enjoy.  I've also put a copy on our gopher site.
Here's the info about obtaining this protocol via gopher:

Gopher to gopher.uoknor.edu

Arrow down to number 5 and hit return:
 -->  5.  Other OU Gophers, Services & Data/

then, arrow down to number 8 and hit return:
 -->  8.  Oklahoma University Genetic Computer Group (OUGCG)/

and then arrow down to number 8 and hit|`it return to view the protocol
 -->  8.  diatom_earth_prep.txt [18-Feb 06:43:36, 23KB].

and then type 'm' to get it mailed to you.

Please reference:
K. Iyer, J. Crabtree, S. Clifton, S. Chissoe, E. Mardis and B. A. Roe, 
(1994) Isolation Of Cosmid, Plasmid and P1 DNAs via Diatomaceous Earth-based
Maxi, Midi and Mini-preps, *and* the non-Diatomaceous Earth-based Mini-prep.
manuscript submitted for publication.

Other relevant references are included at the end of this file.

Bruce A. Roe	  University of Oklahoma	          February 17, 1994
e-mail: broe at aardvark.ucs.uoknor.edu

----------------------------------------------------------------------------
------------------- include file diatom_earth_prep.txt  -------------------

Roe Lab           University of Oklahoma                  February 17, 1994

     Isolation Of Cosmid, Plasmid and P1 DNAs via Diatomaceous Earth-based
     =====================================================================
  Maxi, Midi and Mini-preps, *and* the non-Diatomaceous Earth-based Mini-prep.
  ===========================================================================

I. Diatomaceous Earth-based Maxi-prep
   ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^

A. Materials:
   ^^^^^^^^^

1)  LB (Luria Bertani) media 

	10g Bacto Tryptone
	5g Bacto Yeast extract
	10g NaCl 
              {
	Dissolve the above components in double distilled water . Adjust the 
	volume to a liter and autoclave at 121oC / 15 lbs for 20mins. Cool the 
	solution and add the appropriate amount of antibiotic just before use.
	Typically Amp at 25 ug/ml or Kan at 50 ug/ml.

2)  TB ( Terrific Broth ) media

	12g Bactotryptone
	24g Bacto Yeast extract
	4ml Glycerol

	Dissolve the above components in approximately 900ml double distilled
	water. Autoclave this solution for 20mins at 121oC at 15 psi.  Let the 
	solution cool, add 100ml of TB Salt Solution (described below), and
	adjust the final volume to 1000ml by adding double distilled water.
	Add antibiotic, typically Amp at 25 ug/ml or Kan at 50 ug/ml just prior
	innoculation with cells.

	10 X TB Salt Solution:	For total volume 100ml

	0.17M KH2PO4	2.31g  KH2PO4
	0.72M K2HPO4	12.54g K2HPO4

	Adjust the volume to 100ml with double distilled water and autoclave.

3)  GET / LYSOZYME solution :

	Make this solution fresh or filter sterilize before storing at 4oC.

	For total volume 100ml:

	50 mM Glucose		0.9 g  d-Glucose
	10 mM  EDTA, pH 8.0	1 ml of 1M stock
	25 mM Tris-HCl,pH 8.0	2.5ml of 1M stock

	Make the volume to 100ml with double distilled water. 
	Filter sterilize the above solution if being stored at 4oC.
	
	Add 2mg/ml Lysozyme (0.2 gms lysozyme/100 ml 1xGET) just before use.

4)  NaOH/SDS (Alkaline lysis solution):

	Make this solution fresh

	For total volume 100ml:

		0.2 N  NaOH	20ml of 1N stock
		1% SDS		5 ml of 20% SDS

	Make the volume to 100ml with double distilled water.

5)  3.0 M Sodium Acetate (NaOAc), pH 4.8

6)  DNA binding matrix 

	Weigh 10g of diatomaceous earth (Sigma D-5384) and resuspend it in 
	100ml of double distilled water in a 100ml measuring cylinder and let 
	it settle down for 3 hours. Decant the supernatant. If you wish to
        store the de-fined matrix, resuspend the pellet in TE:10:1, transfer
        the matrix to a screw cap storage bottle and store in the cold room.
        Otherwise, resuspend the pellet in 100ml of 6M guanidine hydrochloride
	containing 50mM Tris-HCl and 20mM EDTA ( final matrix concentration of
	100mg/ml assuming no significant losses during the defining step ).

7)  Wash Buffer (TE/ethanol)

	For total volume 1000ml:

	10 mM Tris-HCl, pH 8.0	10ml of 1M stock
	1 mM EDTA, pH 8.0	1ml of 1M stock
	50%  Ethanol		500ml of absolute alcohol

	Make the volume to 1000ml with double distilled water.

8)  Elution Buffer (TE:10:1) - Note that this is Tris-EDTA:10:1 not the
			       usual 10:0.1 molar ratio of Tris-EDTA.

	For total volume 1000 ml:

	10 mM Tris-HCl, pH 8.0	10ml of 1M stock
	1 mM EDTA, pH 8.0	1ml of 1M stock

	Make the volume to 1000ml with double distilled water.

9)  Ethanol Acetate

	95% Ethanol	95ml of absolute ethanol
	0.12M NaOAc	4ml of 3M NaOAc, pH 4.8.

	Make the volume to 100ml with double distilled water.

10) Isopropanol

11) Acetone

12) 70% Ethanol

13) a)	DNase free RNase A

	For 100ml of RNase A buffer:

	0.1M Na acetate	0.82g anhydrous
	0.3mM EDTA	0.3ml of 100mM stock

	Adjust pH to 4.8 with acetic acid. Make the volume to 100ml with 
	double distilled water.  Aliquot in 2ml amounts and store at -20oC.
	Add 40mgs of pancreatic RNase A to the 2ml alaquots of RNAse A
	buffer just before use and inactivate any contaminating DNase by
        heating at 80oC for 10mins.

    b)	RNase T1 (10 units/ul) made in 50mM Tris-HCl pH 7.6.


B. Methods:
   ^^^^^^^

1)  Grow 2 liters of cells containing desired plasmid, cosmid, P1 chimeric 
    vectors in the following manner:

    * Inoculate cells into 3ml of LB media containing the appropriate 
      antibiotic and grow for 8 hours at 37oC with shaking.

    * Pour the above 3ml culture after 8 hours into 50ml of LB media 
      containing the appropriate antibiotic and grow for 8 hours at 37oC
      with shaking.

    * Split the above 50 ml culture into two aliquots. Pour 25ml aliquots
      each into a 2 liter flask containing 1 liter of LB media with
      appropriate antibiotic. Grow the two liter media ( two flasks )
      at 37oC for 8 hours with shaking.

2)  Pellet the cells from the above two liter culture by centrifugation at
    5 K for 10 mins. Cells may be frozen at this point.

3)  Let the cells thaw at room temperature. Resuspend the cells in 35ml of 
    GET / LYSOZYME solution with the help of a spatula. (Caution: Do not 
    vortex at any time during the procedure because you may shear the 
    chromosomal as well as the cosmid DNA ).

4)  Add 70 ml of ALKALINE LYSIS solution. Mix it gently with a spatula 
    and incubate on ice for 5mins. The solution becomes very viscous and 
    stringy. 

5)  Add 52.5 ml of 3M NaOAc, pH 4.8. Mix gently with a spatula and 
    incubate on ice for 60mins.

6)  Remove precipitated SDS, protein and chromosomal DNA by filtering 
    through a double layered cheesecloth and centrifugation at 12K for 
    10mins at 4oC (twice if required).

7)  To the clear supernatant add DNase free RNase A (final concentration of 
    RNase A in the cleared lysate ~ 40 ug/ml ) and RNase T1 (40 units/ml). 
    Incubate at 37oC for 30mins. 

8)  Add an equal volume of ISOPROPANOL to the clear RNase treated  
    lysate. Incubate at R.T for 5mins and spin the DNA pellet down.

9)  Resuspend the pellet in 1xTE (20-30ml = X). Add 2X volume of 
    100mg/ml matrix. Allow the DNA to bind for 5mins. Spin the DNA 
    bound matrix down.

10) Decant the supernatant and wash the matrix pellet with 2X volume of 
    WASH BUFFER.

11) Decant the supernatant and wash the pellet with 2X volume of 
    ACETONE.

12) Decant the supernatant and dry the pellet in the vacuum oven and/or at 
    65oC until dry .

13) Elute the DNA from the dried pellet with 1X volume of ELUTION 
    BUFFER at 65oC for 10 mins with intermittent stirring.

14) Spin the pellet down by centrifugation at 12K for 10mins and collect 
    the supernatant.

15) Repeat steps 13) and 14) for increased yield of plasmid, cosmid,
    or P1 vector.

16) Combine the supernatants and add 2.5 volumes of ETHANOL ACETATE to 
    precipitate the DNA.

17) Wash the precipitated DNA with 1X volume of 70% ETHANOL and 
    dry the pellet under vacuum.

18) Resuspend the DNA pellet in 2ml of ELUTION BUFFER.

Note: 	Typical average yield:
		P1 or Cosmid:  0.2 - 2ug per ml culture.
		Plasmid (pUC or related clones):  2 - 4ug per ml culture.


II. Diatomaceous Earth-based Midi-prep
    ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^

	Note:   This procedure is the method of choice for isolating
		double stranded plasmid-based templates for the Sequenase
		Dye-Labeled Terminator Sequencing Reactions.

1)  Pick one colony into 3 ml of 2xTY containg the appropriate antibiotic 
    (typically Amp at 25 ug/ml for pUC-based plasmids) and incubate at 37oC
    with shaking for 8-9 hours. 

2)  Transfer seed culture to 50 ml media and incubate as above for 11-14 
    hours.

3)  Harvest cells by transferring into a 50 ml sterile conical tube and 
    spinning at 3000 rpm in GPR tabletop centrifuge for 5 min.  Decant 
    supernatant and freeze cell pellet at -70oC for at least 1 hour.

4)  Resuspend defrosted cells in 2 ml GET/lysozyme (2 mg/ml).  Add 4 ml 
    of NaOH/SDS and incubate on ice for 5min.

5)  Add 4 ml of 3M NaOAc, pH4.8, and incubate on ice for 60min.

6)  Filter the supernatant through cheesecloth into another 50 ml conical 
    tube and spin at 3000 rpm in the GPR centrifuge for 20min.

7)  Decant supernatant to 50 ml polypropylene tube and add 20 mg/ml of 
    DNase-free RNaseA.  Incubate at 37oC for 30 minutes.

8)  Add 7 ml (equal volume) of matrix (20mg/ml in guanadine HCl dissolved
    in 10:1 TE). Mix about 5min.  Spin 5min at 3000 rpm in GPR tabletop 
    centrifuge.

9)  Wash with an equal volume of wash buffer.  Spin.  Decant  supernatant.

10) Wash with an equal volume of acetone.  Spin.  Decant supernatant.

11) Dry in vacuum oven (about 15-20min).

12) Add 600 ul of 10:1 TE, incubate for 10min at 65oC with intermittent 
    mixing.

13) Spin at 8000 for 5min.  Decant supernatant to Eppendorf tube.

14) Spin again and transfer to Eppendorf tube (about 250 ul).

15) Precipitate with 2.5 volumes of ethanol/acetate, rinse with 80% ethanol,
    and dry in a Speed-vac.

16) Resuspend in 40 ul of 10:0.1 TE and assay concentration using 2 ul 
    on an agarose gel.  The yield should be ~1 ug/ml of starting cells.  
    Use ~5 ug in the Sequenase Dye-Labeled Terminator Sequencing Reactions.


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:
   ^^^^^^^^^
1)  TB ( Terrific broth ) media:
	
	For total volume 1000ml

	12g Bactotryptone
	24g Bacto Yeast Extract
	4ml Glycerol

	Dissolve the above in double distilled water and adjust the volume to 
	900ml. Autoclave the solution for 20mins at 121oC /15 lbs. Let the 
	solution cool and then add 100ml of salt solution (described below). 
	Add antibiotic just before use.

	10 X TB Salt Solution:	For total volume 100ml

	0.17M KH2PO4	2.31g  KH2PO4
	0.72M K2HPO4	12.54g K2HPO4

	Adjust the volume to 100ml with double distilled water and autoclave.


2)  GET / Lysozyme solution:

	Make this solution fresh or filter sterilize before storing at 4oC.

	For total volume 100ml:

	50 mM Glucose		0.9 g  d-Glucose
	10 mM EDTA, pH 8.0	1 ml of 1M stock
	25 mM Tris-HCl,pH 8.0	2.5ml of 1M stock

	Make the volume to 100ml with double distilled water. 
	Filter sterilize the above solution if being stored at 4oC.
	
	Add 2mg/ml Lysozyme just before use .

3)  NaOH/SDS ( Alkaline Lysis solution ):
	
	For total volume 10ml

	1% SDS		0.5ml of 20% stock
	0.2N NaOH	2ml of 1N stock

4)  3.0 M Sodium Acetate (NaOAc), pH 4.8

5)  DNA binding matrix

	Weigh 5g of diatomaceous earth (Sigma D-5384) and resuspend it in 
	50ml of double distilled water in a 100ml measuring cylinder and let 
	it settle down for 3 hours. Decant the supernatant. If you wish to
	store the de-fined matrix, resuspend the pellet in TE:10:1, transfer
	the matrix to a screw cap storage bottle and store in the cold room.
	Otherwise, resuspend the pellet in 250ml of 6M guanidine hydrochloride
	containing 50mM Tris-HCl and 20mM EDTA ( final matrix concentration of
	20 mg/ml assuming no significant losses during the defining step ).

6)  Wash Buffer (TE/ethanol)

	For 100ml total volume

	10 mM Tris-HCl, pH 8.0 	1ml of 1M stock
	1 mM EDTA, pH 8.0	0.2ml of 0.5M stock
	50%  ethanol		50ml of absolute alcohol
	Adjust the volume to 100ml with double distilled water.

7)  Elution Buffer (TE:10:1) - Note that this is Tris-EDTA:10:1 not the
                               usual 10:0.1 molar ratio of Tris-EDTA.

	For total volume 100ml

	10 mM Tris-HCl, pH 8.0	1ml of 1M stock
	1 mM EDTA, pH 8.0	0.2ml of 0.5M stock
	Adjust the volume to 100ml with double distilled water.

8)  Ethanol Acetate

	For total volume 100ml

	95 %  Ethanol			95ml of absolute alcohol
	0.12M sodium acetate, pH 4.8	4ml of 3M sodium acetate, pH 4.8
	Adjust the volume to 100ml with double distilled water.

9)  Isopropanol

10) 80%  Ethanol

11) 1XTE - This is the usual TE:10:0.1 used for dissolving nucleic acids.

	For total volume 100ml

	10mM 	Tris-HCl, pH 8.0	1ml of 1M stock
	0.1mM 	EDTA, pH 8.0		20uls of 0.5M stock
	Adjust the volume to 100ml with double distilled water.


12) a)	DNase free RNase A ( 20 ug/ul )

	For 100ml of RNase A buffer:

	0.1M Na.acetate	0.82g anhydrous
	0.3mM EDTA	0.3ml of 100mM stock
	
	Adjust pH to 4.8 with acetic acid. Make the volume to 100ml with 
	double distilled water.  Aliquot in 2ml amounts and store at -20oC .  
	Add 40mgs of pancreatic RNase A to 2ml RNaseA buffer just before use
	and inactivate any contaminating DNase by heating at 80oC for 10mins.
	yielding a 20 ug/ul RNase A stock.

    b)	RNase T1 (10 units/ul ) made in 50mM Tris-HCl pH 7.6 stock

13) 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.

9a) 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.

9b) 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.

IV. References.

1. Birnboim, H.C. and J. Doly. 1979.  A rapid alkaline extraction procedure for
screening recombinant plasmid DNA. Nucl. Acids Res. 7:1513-1523. 
2. Pouwels, P.H.,  C.M. Knijnenburg, J. van Rotterdam and J.A. Cohen. Structure
of the replicative form of Bacteriophage. 1968. J.Mol.Biol.  32: 169-182.
3. Britten, R.J., D.E. Graham and B.R. Neufeld. 1974. Analysis of repeating DNA
sequences by reassociation. Methods in Enzymology (Wu, R., Grossman, L. and
Moldave, K., Eds. )29: 363-418. 	
4. Birnboim, H.C. 1983. A rapid alkaline extraction method for the isolation of
plasmid DNA. Methods in Enzymology  (Wu, R., Grossman, L. and Moldave, K.,
eds.) 100: 243-255.
5. Clewell, D.B., and D.R. Helinski. 1969. Supercoiled circular DNA-protein
complex in E.coli: Purification and induced conversion to an open circular DNA
form. Proc. Natl.Acad.Sci.U.S.A. 62:1159-1166. 
6. Serghini, M.A., C. Ritzenhaler and L. Pinck. 1989. A rapid and efficient
'miniprep' for the isolation of plasmid DNA. Nucl. Acids. Res. 17: 3604.
7. Johnson, R.K. 1990. A small scale plasmid preparation yielding DNA suitable
for double stranded sequencing and in Vitro transcription. Anal.Biochem. 190:
170-174.
8. Chowdhury, K. 1992. One step miniprep method for the isolation of plasmid
DNA. Nucl.Acids.Res. 19: 2792.
9. Colman, A., M.J. Byers, S.B. Primrose and A. Lyons. 1978. Rapid purification
of plasmid DNAs by Hydroxyapatite chromatography. Eur.J.Biochem. 91: 303-310.
10. Sparks, R.B. and J.H. Elder. 1983. A simple and rapid procedure for the
purification of plasmid DNA using reverse-phase C18 silica beads. Anal. 
Biochem. 135:345-348.
11. Micard, D., L.M. Sobrier, J.L. Couderc and B. Dastugue. 1985. Purification
of RNA free plasmid DNA using alkaline extraction followed by Ultrogel A2 column
chromatography. Anal.Biochem. 148: 121-126. 
12. Marko, M.A., R. Chipperfield and H.C. Birnboim. 1982. A procedure for the
large scale isolation of highly purified plasmid DNA using alkaline extraction
and binding to glass powder. Anal.Biochem. 121: 382-387. 
13. Vogelstein, B. and D. Gillespie. 1979. Preparative and analytical
purification of DNA from agarose. Proc.Natl.Acad.Sci. 76: 615-619.
14. Boom, R., C.J.A. Sol, M.M.M. Salimans, C.L. Jansen, P.M.E. Wertheim-van
Dillen and J. van der Noordaa. 1990. Rapid and simple method for purification
of nucleic acids. Jour. of Clin. Micro. 28: 495-503.
15. Voskuil, I.M. and H.G. Chambliss. 1993. Rapid isolation and sequencing of
purified plasmid DNA from Bacillus subtilis. Applied and Environmental
Microbiology. 59: 1138-1142.
16. Carter, J.M. and I.D. Milton. 1993. An inexpensive and simple method
for DNA purifications on silica particles. Nucl.Acids.Res. 21: 1044.
17. Willis. E.H., E.R. Mardis, W.L. Jones and M.C. Little. 1990. Prep-A-GeneTM:
A superior matrix for the purification of DNA and DNA fragments. Biotechniques
9:92-99.
18. Hall. R.H. 1967. Partition chromatography of nucleic acid components
(Isolation of the minor nucleosides). Methods in Enzymology ( Grossman. L. and
Moldave. K. eds.) 12A:305-315.
19. Volcani, B.E. 1981.Silicon and Siliceous structures in biological systems.
(Simpson .T.L. and B.E.Volcani eds.).Springer-Verlag, New York: 157-200.
20. Woodcock, D.M., P.J. Crowther, J. Doherty, S. Jefferson, E. Decruz, M.
Noyer-Weidner, S.S. Smith, M.Z. Michael and M.W. Graham. 1989.  Quantitative
evaluation of Escherichia coli  host strains for tolerance to cytosine
methylation in plasmid and phage recombinants.Nucl.Acids. Res. 17: 3469-3478.
21. Bullock, W.O., J.M. Fernandez and J.M. Short. 1987. XL1-Blue: A high
efficiency plasmid transforming recA Escherichia coli strain with
beta-galactosidase selection. Biotechniques. 5: 376-378.
22. Taylor. R.G., D.C. Walker and R.R. McInnes. 1993. E.coli host  strains
significantly affect the quality of small scale plasmid DNA preparations used
for sequencing. Nucl. Acids. Res. 21:1677-1678.
23. Cross. S.H. and P.F.R. Little. 1986. A cosmid vector for systematic
chromosome walking. Gene 49: 9-22.
24. Pierce. J.C., B. Sauer and N. Sternberg. 1992. A positive selection vector
for cloning high molecular weight DNA by the bacteriophage P1 system: Improved
cloning efficiency. Proc. Natl. Acad. Sci. USA 89: 2056-2060.
25. Sambrook, J., E.F. Fritsch  and T. Maniatis. 1989. Molecular Cloning: A
laboratory manual. Cold Spring Harbor Press, NY. 
26. GS Gene Prep Manifold Instruction Manual, BioRad Labs, Richmond, CA.
27. Chen, E.Y. and P.H. Seeburg. 1985. Supercoil sequencing : A fast and
simple method for sequencing plasmid DNA. DNA 4:165-170.

-------------- End of diatom_earth_prep.txt February 17, 1994 --------------