Dr. Duncan Clark
duncan at genesys.demon.co.uk
Wed Apr 1 09:21:44 EST 1998
In article <188.8.131.5280331102109.006d5e18 at imm2.imm.uth.tmc.edu>, David
L. Haviland, Ph.D. <dhavilan at IMM2.IMM.UTH.TMC.EDU> writes
>Basically, I was considering getting my own 'wizzard' style prep going and
>through the years I've inheritied two bottles of the Silica, having known
>individuals that claim this works. I have not yet tried it 'in my own hands'.
Try the following as published on this newsgroup maybe a couple of years
or so ago. I can email it as an MS word file if that is easier to read
given the lousy cut and paste formatting below:
In our hands it works very well.
The following protocol is based on using Guanidine hydrochloride-Celite
resin slurry and uses the same reagents as the Magic Miniprep kit. A
strong vacuum suction apparatus with a collection trap is required.
1M Tris.HCl, pH 7.5; 0.5M EDTA, pH 8.0; 2M NaOH; 10% SDS Potassium
Acetate; Glacial Acetic Acid; Guanidine hydrochloride (purum; >98%)--- -
- Fluka catalog 50950 --- 1kg = $28.00 Diatomaceous Earth Flux
calcined (need not be Acid washed) (Celite( 545) Available from :-
Fluka catalog 22140 ---- 1kg = $13.90 5 kg = $44.90 Also available
in bulk from Celite Corp., P.O. Box 519 Lompoc, California 93438- 0519,
U.S.A. (Tel: (805) 735-7791; Fax: (805) 735-5699 ) 50 lb bag of Flux
calcined Celite 545 = $22.00. Contact them for info. about the
distributor in your area RNAseA (DNAse free --Boehringer Mannheim)
Magic Miniprep Minicolumns (Promega)
Buffers & Solutions :-
Merlin I (Also known as Cell resuspension soln or Soln.1 of PromegaUs
50mM Tris.HCl, pH 7.5; 10mM EDTA; 100 ug/ml RNAseA (DNAse free)
Merlin II (Also known as Cell Lysis Soln. or Soln. 2 of PromegaUs kit)
0.2M NaOH; 1% SDS
Merlin III (Also known as Neutralization Soln. or Soln.3 of PromegaUs
To make 500 ml. Dissolve 61.35 gm of solid potassium acetate and 35.7 ml
of glacial acetic acid to make a final volume of 500 ml in MilliQ H2O.
Merlin IV ( Celite resin slurry in 7M Guanidine hydrochloride buffer;
preparation was secret till now) :- To make 100 ml of resin slurry :-
Dissolve 66.84 gm of Guanidine hydrochloride in 33.333 ml of Merlin III
buffer (composition given above). Stir in a very clean 250 ml glass
reserved solely for this purpose. Use a magnetic stirrer with gentle
heating. Most of the guanidine hydrochloride will dissolve in 5-10 min.
there will be a fine fluffy preciptate that wont dissolve right now.
pH to 5.5 using a 0-14 pH indicator paper and 10M NaOH. You will need
2.0 ml of 10M NaOH for this. pH adjustment need not be ultra-precise,
level of accuracy obtained with the pH paper will do. Continue stirring
during pH adjustment. Pour the contents of the beaker into a clean 100
measuring cylinder reserved for this purpose only. The volume will be
70-80 ml. Add MilliQ H2O to make upto 100 ml. and pour back into beaker
and continue stirring with gentle heat for another 5 min.
Most of the fluffy precipitate present earlier will dissolve. Meanwhile
weigh out 1.5 gm of Celite powder into a very clean glass bottle
for this purpose only. Filter the guanidine hydrochloride solution in
beaker thru 1 layer of whatman filter paper using a clean filter funnel
collect the filtrate directly into the bottle containing the Celite
Shake very well to make a slurry and store at room temperature until
Merlin V (Also known as Column Wash Soln. of PromegaUs kit) :- 200mM
NaCl; 20mM Tris.HCl, pH 7.5; 5mM EDTA; 50% Ethanol Note:- It is
important that the this soln. contain at least 20% of a lower alcohol.
50% Methanol or 50% Isopropanol may be substituted for Ethanol. Use of
alcohols higher than
Isopropanol (eg. Butanol) will result in residual alcohol remaining in
resin since these alcohols are less volatile and will not be easily
TE Buffer :-
10mM Tris.HCl, pH 7.5; 1mM EDTA
MiniPrep Protocol :-
The following steps describe a standard plasmid miniprep starting with a
3ml overnight LB culture.
1. Spin down 3ml of bacterial culture in a microfuge tube (YouUll have
to do 2
spins of 1.5 ml each in the same tube) and discard the supernatant.
2. Resuspend the bacterial pellet in 200 ul of Merlin I soln.
3. Add 200 ul of Merlin II soln. to the resuspended bacterial
suspension will clear almost immediately on addition of Merlin II soln.
it does'nt, then mix by inversion a few times.
4. Add 200 ul of Merlin III soln., vortex for 2-3 sec. and spin at
14,000 x g
for 5 min. at room temp.
5. Prepare a fresh tube (identical label) with 1ml of Merlin IV resin
Shake resin bottle well to resuspend before use. Use a P-1000 tip with
tip sliced (to increase the bore) to aspirate the slurry into the fresh
6. Aspirate the supernatant in the tube from step 4 (600 ul volume) and
transfer into the fresh tube containing the resin slurry. You may mix
inversion but this is not necessary.
7. Assemble a vacuum line with a tube capable of snugly fitting a Magic
mini-prep column (available from Promega at $110/- for 250 minicolumns;
estimated cost of manufacture = $25/- or less for 250 columns).
8. Load the resin-DNA slurry onto the column and allow the resin bed to
formed under constant suction. Save the microfuge tube for step 10.
9. Screw on a 3ml luer-lock syringe onto the column and load 2 ml of
soln. and allow washing of the resin bed to proceed.
10. Transfer the column back into the original microfuge tube that
the resin- DNA slurry. The column will fit very tightly and must be
into the tube. Spin the tube for 20 sec. to dry the column thoroughly.
11. Transfer the column into a fresh (identical labelled) microfuge tube
load 50 ul of pre-heated (70oC) TE. You may use water but I prefer TE
since the DNA is more protected from chance nuclease digestion due the
presence of the EDTA in TE.
12. Spin the column in the microfuge tube for 30 sec to elute the DNA.
column may be then discarded. The DNA eluted from it and collected in a
50 ul volume in the bottom of the microfuge tube is now ready for
restriction digestion or sequencing.
NOTE:- If you do not have a vacuum setup, you may use the Promega
minicolumns connected to a luer lock syringe as a push column in steps 8
& 9. Be sure to disconnect the syringe from the column between steps 8
& 9 before pulling back on the plunger.
Reagents Required :-
Buffer I :- 50mM Glucose; 25mM Tris.HCL, pH 8.0; 10mM EDTA pH 8.0
Buffer II:- 0.2M NaOH; 1% SDS (Same as Merlin II soln.)
Buffer III:- Same as Merlin III soln.
MerlinMax Resin Slurry:- Same as Merlin IV resin slurry except that
add 15gm of Celite powder to 100 ml of 7M Guanidine hydrochloride
MerlinMax Binding buffer :- Prepare 100 ml of 7M Guanidine
buffer solution as described for the Merlin IV resin slurry earlier.
BUT DO NOT ADD ANY CELITE POWDER.
Merlin V Soln. :- Described earlier.
Econo-Pac Columns (Biorad catalog 732-1010, 50 columns = $80/-)
Merlin Maxi-Prep Protocol:-
The following describes a standard alkaline lysis maxi-prep protocol of
250ml overnight Terrific Broth bacterial culture.
1. Spin a 250ml TB broth overnight culture in a 25o ml Sepcor centrifuge
bottle at 6,000 rpm x 10min x 4oC in a Beckman JA-14 rotor.
2. Decant supernatant and resuspend the bacterial pellet in 40ml of
3. Add 80ml of buffer II, swirl and keep on ice for 5 min. exactly.
4. Add 40ml of buffer III, mix by shaking and keep on ice for at least
5. Spin in a JA-14 rotor at 8,000 rpm x 10min x 4oC.
6. Filter supernatant thru one layer of Scotsman paper towel (white) or
layers of Kimwipes tissue paper and collect the filtrate in a fresh
7. Add isopropanol to make volume upto 250 ml. (ie. 90 ml or > 0.6 x
volume of filtrate). Shake well to mix and spin in a JA-14 rotor at
12,000 rpm x
15min x 4oC.
8. Decant supernatant and rinse crude DNA pellet with 70% ethanol,
decant and allow the DNA pellet to dry by inversion of the bottle over
a paper towel.
9. Dissolve the DNA pellet in 4ml of TE buffer.
10. Aliquot 10ml of MerlinMax Resin Slurry into a 50ml sterile tube and
40ml of MerlinMax Binding buffer to it.
11. Add the 4ml of DNA solution in TE obtained from step 9 to the Resin
Binding buffer mixture in step 10. Mix with gentle shaking on a rocker
platform for 20-30 min.
12. Shake the Resin slurry-DNA mix well and load onto an Econo-Pac
connected to a vacuum line under constant suction. Allow the resin bed
form under vacuum.
13. After all of the buffer has been sucked thru and the resin bed is
dry, top off the column with Merlin V soln (about 20ml).
14. Allow washing of the column by suction.
15. After the bed is dry continue suction for additional 5 min. to
traces of ethanol from the column.
16. Disconnect the vacuum line and add 5 ml. of pre-heated 70-80oC TE
buffer to the column. Place the column into a 50 ml tube and spin at
2500 rpm in a Sorvall table-top centrifuge for 5min. at room
17. Transfer the eluate at the bottom of the 50ml collection tube into a
Sarstedt tube. Respin the column for an additional 5min. to collect
residual eluate in the column bed. Transfer the eluate collected in the
2nd. spin to the same Sarstedt tube that received the 1st.
18. Add 0.1 vol. of 3M Sodium acetate and an equal volume of
Vortex and freeze on dry ice for 30 min.
19. Spin at 12,000 x g for 15min. at 4oC to pellet the DNA.
20. Rinse the DNA pellet once in 70% ethanol, dry and dissolve in 500 ul
of TE buffer.
21. Transfer to a 1.5 ml microfuge tube, add 1 ul of RNAseA (DNAse free)
and incubate at 37oC for 30min.
22. Phenol/Chloroform extract the DNA solution, repreciptate it, rinse
70% Ethanol and dry it.
23. Dissolve the final DNA pellet in 500 ul TE. Take A260 & A280
DNA is ready for use.
Note:- The DNA thus obtained is clean enough for transfections.
However an additional precipitation step in CTAB may be performed if
CTAB Preciptation Protocol
1. Add 0.1 vol. of 5% CTAB solution in water (Sigma Cat H 6269) to the
solution. Note:- The solution usually precipitates at room temperature.
Prewarm at 37oC for 30 min. to dissolve before use.
2. Vortex well and allow to stand at room temperature for 10-20 min. The
DNA preciptates on addition of the CTAB.
3. Spin at room temp. (14,000 x g for 10 min). Aspirate supernatant and
dissolve the pellet in 400-500 ul of 1.2 M NaCl. You may have to
up & down for this.
4. Add equal volume of Isopropanol, vortex, freeze and spin (14,000 x g
15 min at 4oC.
5. Rinse DNA pellet twice in 1 ml of 70% Ethanol each time. Air dry and
dissolve DNA in TE. DNA is ready for use.
The typical yields of DNA with the Merlin Mini-prep reagents is
=/> 15.0 ug of DNA. The yields in the Merlin Maxi-preps is =/>2-4mg.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
More information about the Methods