Tk:merlin

Re-POST of THE ORIGINAL MERLIN MINI/MAXI-PREP. Ravi Iyer rriyer at unix1.circ.gwu.edu Sun Apr 17 10:06:25 EST 1994

Dear Netter,

I have been following all the postings regarding the Magic/Wizard/Merlin controversy ever since I started the ball rolling with the first bionet posting on the method of recreating the Magic miniprep resin at home -- which I called as "Merlin Mini/Maxi-preps". I am obliged to the contributions made to my original Merlin post (in Nov'93) by various members of the net community. The protocol for the actual formulation of the Merlin resin was actually done in the last 2-3 wks of my postdoc at Harvard Medical School in Sept.'93 and I posted the protocol on Bionet in Nov.'93. I was rather amused to note the recent submission of a manuscript on the process DNA purification by binding to Diatomaceous earth by an Oklahoma group in 1994 after I had publically released the protocol on the Internet in Nov.'93 and after the process itself has been public domain knowledge since 1991.

However I am also delighted with the worldwide response my original posting has generated. Of special mention has been the efforts by Dr. Chia Jin Ngee of the National University of Singapore (Internet address: "mcblab47 at .leonis.nus.sg") to further refine my original protocol and post the results of the same on the bionet. However I would have been even more delighted if there had been an acknowledgement of the original source of the protocol in each of these postings. In the rather free-wheeling world of Internet, it may be easy to forget such niceties, but I am sure you will agree that as professionals we cannot afford to overlook the most basic etiquette in Sciencenamely the integrity of proper attribution of sources.

The following protocol has undergone further modifications since its initial conception. The Fluka Celite has been found to be erratic in its yield. By contrast the Celite avaliable from Sigma has been much more consistent. It is possible that you may have to test several grades of Celite from Fluka before deciding on the best one.

The following is the summarrized data on various resins collected by Dr. Chia Jin Ngee (mcblab47 at leonis.nus.sg):-

The various resins are ordered in descending order of performance:-

1a. Kieselgel 60 (Merck)                  ] 1b. Diatomaceous Earth (Sigma D 5384)     ]---These 3 appear to be the best 1c. Celite for Analytical Filtration (BDH) ] 2. Silica Gel for TLC (Aldrich) 3a. Kieselgel 60H (Merck) 3b. Celite 545 (Fluka) 4. Kieselgel 40 (Merck)

Best of luck. Dr. Ravi R. Iyer, MD, Ph.D. Dept. of Medicine, George Washington University Medical Center, Washington DC

Merlin DNA Prep RESIN

One of the developments in Molecular Biology in the past 5 years has been the proliferation of kits that enable almost any one with some biology experience to clone from a box. While such kits definitely save time and energy, they also tend to give rise to a kit generation of biologists who are gradually programmed to implicitly accept every product put forth by companies. The current pressures on graduate students, postdocs and faculty to publish or perish also tends to promote the reliance on these kits. Last but not least, the entry into the field of a growing number of professionals from other areas of biology and medicine who wish to use the tools Molecular Biology but do not have the time to learn it from scratch is an additional factor that provides impetus to this trend. Most kits do provide immense value, however amidst this crowd are a number of recent arrivals that while useful are being marketed at exhorbitant markups. The scenario shares an uncanny resemblance to the current debate over the cost of drugs being marketed by pharmaceutical companies. One such area are the technologies associated with the small scale purification of DNA (plasmid or genomic) and PCR fragments in the lab. The average lab performs about 100-200 mini-preps, 5-10 CsCl2 maxi-preps and 25-50 PCR reactions a week. The current alkaline-lysis/Phenol-Chloroform miniprep costs about $1.17 a prep, while the alkaline-lysis/CsCl2 maxi-prep costs about $22/- a prep. This translates into a per week recurring cost of about $650/- in reagents and consumables. This does not include the costs of hazardous waste disposal, depreciation costs of capital outlay in ultracentrifuge equipment, service contracts for equipment maintenance, the human costs involved in the handling of toxic chemicals and the time consumed by the standard procedures. The recurring nature of these processes as well as the high cost of currently used processes makes resin-based DNA purification kits a biotech. marketers bonanza. What the scientific community has been ignorant about is that the information about these technologies is freely available in the public domain for several years. The cost of manufacturing these reagents is incredible small and no more skill than the ability to measure liquids and weigh solids is required. Our ignorance about them contributes to the cost of research and siphons valuable dollars away from more worthy investments. The following section describes a protocol for a silica based DNA purification resin. I have chosen to call my protocol as --- Merlin Mini-preps ---, but this name is purely a facetious choice. The main thing is that it works very well. After all --- A rose if called by any other name -.

Merlin DNA Purification System

Background History:

The following protocol is based on the fact that Silica & silcates like Sodium-silicate (Glass beads or Glass milk) will bind DNA (single-stranded, double stranded, genomic or plasmid) in the presence of a suitable chaotrope. The original observation was made with respect to the binding of DNA to glass in the presence of a chaotrope such as Sodium iodide, Sodium perchlorate or Sodium trichloroacetate. This led to the marketing of glass milk based DNA purification products (GeneClean, Sephaglass etc). However the DNA recovery was typically low and yields were erratic. In Dec.1991, BioRad was granted a patent on the use of diatomaceous earth, which is 99% silicon dioxide (SiO2) for the purpose of DNA purification. Diatomaceous earth (or CELITE) has been in use as a non-specific adsorbent and filter-aid for over 30 yrs., and is the sedimentous remains of the cell walls of microscopic marine organisms or diatoms. The process claimed by BioRad in their patent involved the use of a slurry of Celite suspended in 7M chaotrope solution in which the chaotrope could be any one of the following --- Sodium iodide, Sodium perchlorate or Sodium trichloroacetate. Within one month of issue of Biorad's patent, Promega Corp. began marketting a diatomaceous earth based DNA purification product called Magic Mini-preps. The resin used in Magic Minipreps is a Celite slurry suspended in 7M Guanidine Hydrochloride-1M KOAc buffer, pH 5.5. Promega very cleverly bypassed the claims of Biorads patent by using a Celite slurry suspended in 7M Guanidine Hydrochloride which was not specified by Biorad as a suitable chaotrope in their patent. In addition, Promega also circumvented the required use of 50% Ethanol (50% Methanol or 50% Isopropanol is equally suitable) in the wash buffer, by shipping the Magic Mini-prep kit minus the Ethanol and requesting the customer (or end-user) to add the Ethanol himself. The reason they gave was that special shipping licenses are required for solutions containing high percentages of alcohol, but in effect this ploy afforded them the dual protection of transferring the onus of infringement of the Biorad patent onto the customer.

NOTE: ( Promega recently modified their original Celite resin -- or Magic miniprep resin and are currently selling a derivatized Celite under the name of Wizard. However the bionet network was recently swamped with complaints from users worldwide that the Wizard resin was not working or was producing erratic results).

The use of Celite as a DNA purification resin is enormously advantageous. Celite of the appropriate grade binds upto 2-3 mg of DNA per gm of resin which is far in excess of the amounts required for experimental purposes. In addition the reagents used are non-toxic and eliminate hazardous waste disposal. The process is 2-3 times as fast and the DNA obtained is of high purity. Finally the cost-savings are tremendous. The average lab performs about 100-200 mini-preps, 5-10 CsCl2 maxi-preps and 25-50 PCR reactions a week. The standard alkaline-lysis/Phenol/chloroform method costs about $1.17 per prep in reagents and plasticware plus additional disposal costs of phenol & chloroform and is also toxic and tedious. By contrast the standard Celite mini-prep costs 55 cents in reagents and plasticware with no additional costs and is fast with high quality DNA as the result. The standard alkaline lysis/CsCl2 spun maxi-prep costs $22/- in reagents and plasticware plus additional depreciation costs of the ultracentrifuge use and the service charge of maintaining the instrument. Also to be added are the costs involved in Ethidium bromide disposal as well as the toxic hazard involved and the fact that it typically takes about 24-36 hrs. to obtain the DNA. By contrast the Celite method costs $5/- per maxiprep in reagents and plasticware with no additional costs and the DNA is available 4 hrs. after commencement of the alkaline lysis with yields upto 2-4 mg of high grade DNA. All this translates into a per week recurring cost of about $650/- in reagents and consumables when using the standard miniprep/maxiprep protocols versus about $200/- using the Celite method. Companies like Promega, market the Celite resin at rates of $440/- per liter which is a cost that is only slightly lower than the cost of the standard protocols when the actual cost of making the resin yourself is only about $30/- per liter and 30 minutes of your time. What is more you can have all those research dollars in your account go towards a more useful/important expense (maybe hiring that extra technician) than paying for the Promega executive jet.

Merlin-MiniPrep Protocol

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. This protocol was developed by me during my fellowship at Childrens Hospt., Boston and is currently being used very successfully there. Feel free to distribute this as widely as you wish. I trust you to appropriately acknowledge its source when you distribute it.

Reagents 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%) Available from:- Fluka catalog #50950 --- 1kg = $28.00

Diatomaceous Earth Flux calcined (need not be Acid washed) or Celite-545 Available from :- Sigma catalog# D 5384 --- 1kg = $548.00 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 ) A 50 lb bag of Flux calcined Celite 545 = $22.00 Contact them for info. about the distributor in your area

Please note that I have tested only the Sigma variety. Theoritically the Celite from Fluka or from other sources should work just as well. However I do know of occasional problems experienced with the other sources of Celite. You may need to test several grades from these sources before deciding on the best and sticking to it.

RNAseA (DNAse free --Boehringer Mannheim) Magic Miniprep Minicolumns (Promega)

Buffers & Solutions :

Merlin I (Also known as Cell resuspension soln or Soln.1 of the Promega kit):-    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 the Promega kit): 0.2M NaOH; 1% SDS #

Merlin III (Also known as Neutralization Soln. or Soln.3 of the Promega kit): 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 beaker reserved solely for this purpose. Use a magnetic stirrer with gentle heating. Most of the guanidine hydrochloride will dissolve in 5-10 min. but there will be a fine fluffy preciptate that won't dissolve right now. Don't worry about it. Adjust pH to 5.5 using a 0-14 pH indicator paper and 10M NaOH. You will need 1.5-2.0 ml of 10M NaOH for this. pH adjustment need not be ultra-precise, the 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 ml measuring cylinder reserved for this purpose only. The volume will be about 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 reserved for this purpose only. Filter the guanidine hydrochloride solution in the beaker thru a 0.45 um filter and transfer the filtrate directly into the bottle containing the Celite powder. Shake very well to make a slurry and store at room temperature until use.

Merlin V (Also known as Column Wash Soln. of the Promega 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% is usually used). You may substitute 50% Methanol or 50% Isopropanol for Ethanol. Use of alcohols higher than Isopropanol (eg. Butanol) will result in residual alcohol remaining in the resin since these alcohols are less volatile and will not be easily removed in vacuo.

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. The suspension will clear almost immediately on addition of Merlin II soln. If 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 slurry. Shake resin bottle well to resuspend before use. Use a P-1000 tip with the tip sliced (to increase the bore) to aspirate the slurry into the fresh microfuge tube.

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 by 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 be 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 Merlin V soln. and allow washing of the resin bed to proceed.

10. Transfer the column back into the original microfuge tube that contained the resin-DNA slurry. The column will fit very tightly and must be pushed 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 and 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. The 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 either 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 stepa 8 & 9 before pulling back on the plunger.

Merlin Maxi-Preps

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 you have to add 15 gm of Celite powder to 100 ml of  7M Guanidine hydrochloride buffer soln.

MerlinMax Binding buffer :-  Prepare 100 ml of 7M Guanidine hydrochloride buffer solution as described for the Merlin IV   resin slurry earlier. BUT DO NOT ADD ANY CELITE POWDER.

Merlin V Soln. :- Described earlier.

TE buffer

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 a 250ml overnight Terrific Broth bacterial culture.

1. Spin a 250ml TB broth overnight culture in a 250 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 buffer I.

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 30 min.

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 2 layers of Kimwipes tissue paper and collect the filtrate in a fresh Sepcor 250ml bottle.

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 add 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 column connected to a vacuum line under constant suction. Allow the resin bed to 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 20 ml).

14. Allow washing of the column by suction.

15. After the bed is dry continue suction for additional 5 min. to remove all 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 temperature.

17. Transfer the eluate at the bottom of the 50ml collection tube into a 14 ml 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 isopropanol 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 with 70% Ethanol and dry it.

23. Dissolve the final DNA pellet in 500 ul TE. Take A260 & A280 readings. 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 desired if superclean-LPS free DNA is required.

CTAB Preciptation Protocol

1. Add 0.1 vol. of 5% CTAB solution in water (Sigma Cat # H 6269) to the DNA 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 pipette up & down for this.

4. Add equal volume of Isopropanol, vortex, freeze and spin (14,000 x g for 15 min at 4oC.

5. Rinse DNA pellet twice in 1 ml of 70% Ethanol each time. Air dry and dissolve DNA in TE (or Pyrogen free water for human irrigation --- available from hospital stores). DNA is ready for use.