Gene Transfer and Expression Protocols

416 Pages·1991·27.589 MB·English

by  Edward J. Murray

Preparation of Recombinant Plasmid
DNA for DNA-Mediated Gene llransfer
R&my A&in, Michael WeinfeZd,
and Malcolm C. Paterson
1. Introduction
Recombinant plasmid constructs are frequently employed in transfec-
tion experiments. With the availability of a wide spectrum of specialized and
versatile eucaryotic cloning/expression vectors, investigators have been
given powerful tools to expedite the elucidation of mechanisms governing
gene expression, and to facilitate the identification of genes participating in
diverse cellular processes (namely, metabolism, the immune response, diE-
ferentiation and development, the repair of DNA damage, and malignant
transformation).
The success of a particular gene transfer experiment depends largely on
the quality of the donor DNA preparation. Indeed, the requirement for highly
purified form I (covalently closed circular supercoiled) plasmid molecules,
to ensure both consistency and optimal levels of gene expression in “tran-
sient” assays,a s well as reproducible frequencies of stable transfection, is well
documented.
Many plasmid purification schemes exist, but the Triton lysis/CsCl
“double banding” procedure is the one most frequently quoted as providing
transfectiongrade DNA. The technique, however, does require a consider-
able amount of time (3-5 d) and relies on specialized ultracentrifugation
From: Methods in Molecular Biology, Vol. 7: Gene Transfer and Expression Protocols
Edited by: E. J. Murray 0 1991 The Humana Press Inc., Clifton, NJ
3
Aubin, Weinfeld, and Paterson
equipment. As a result, only a limited number of large-scale plasmid prepa-
rations can be processed at once. A detailed description of this protocol
was recently published by Gorman (I) and is presented here with only mi-
nor modifications.
By way of an alternative, this chapter also describes how milligram quan-
tities of comparably pure form I plasmid DNA (i.e., free of contaminating
bacterial chromosomal DNA, RNA, and other host cell components) can be
obtained by incorporating the acidified-phenol extraction scheme of Zasloff
and coworkers (2) as a final step to the preparative alkaline lysis procedure of
Birnboim (3). The method is rapid (l-2 d) and lends itself to the preparation
of multiple plasmid stocks. Briefly, plasmid molecules are first extracted from
bacteria at alkaline pH (in the range of 12.0-12.6) in the presence of deter-
gent. Under these conditions, linear (chromosomal) DNA will denature,
whereas intact plasmid duplexes will not. Neutralization of the lysate at high
ionic strength allows essentially all of the chromosomal DNA and most of the
host cellular RNA and protein to precipitate, while the plasmid remains in
solution. Complete removal of residual RNA and protein is then accomplished
through a sequence of LiCl precipitation, RNAse and proteinase K digestion,
phenol extractions, and ethanol precipitations. At this stage, a plasmid
preparation consisting of >90% form I molecules is routinely obtained. Fx-
traction with acidified phenol is then used to remove selectively linear and
open circular duplexes from the preparation in one step. The mechanism
appears to exploit differences in the hydrophilic character of the different
DNA species (4). At pH 4.0 in the presence of 75 mMNaC1, open circular and
linear molecules are converted to their single-stranded forms and are there-
fore less hydrophilic than the form I duplexes, because the bases are not
shielded from the aqueous environment. In addition, the protonation of the
nucleotide bases contributes to reduce significantly the overall charge of the
DNA. As a result, open circular and linear DNA species are partitioned to the
acidified phenolic phase, whereas intact form I plasmid molecules are re-
tained in the upper aqueous layer. One round of acid-phenol extraction is
usually sufficient to obtain a preparation consisting of essentially 100% form
I plasmid DNA. A schematic outline of the alkaline lysis/acid-phenol purifi-
cation method is presented in Fig. 1.
2. Materials
All stock solutions and buffers should be prepared using “nanopure”
(double distilled and deionized) water and chemicals of the highest purity
available (i.e., certified as molecular biology grade). In addition, and unless
stated otherwise, buffers as well as all glass and plasticware should be
autoclaved to inactivate contaminating deoxyribonucleases.
Plasmid DNA Preparation 5
Transformed bacterial strain
5 ml sterile LB-broth t antibiotic Grow overnight
0
Innoculate 250 ml to 1 L Grow until O.D.aee reaches 0.8
sterile LB-broth t antibiotic Add chloramphenicol (or spectinomycin)
Continue growth for 20 hours
Harvest/wash cells
Lysis (PEB I, II, III)
500 ml (250 ml) Oak Ridge bottle(e) Precipitate plasmid DNA and bacterial RNA (twice)
a
0
Bulk removal of bacterial RNA (LiCl-MOPS/heating)
30 ml Oak Ridge tube(s) a Ethanol precipitation of plasmid DNA (twice)
u
RNAse A/T1 digestion
Proteinase K digestion
30 ml (15 ml1 Corex tube(s) Phenol extzactions
a Rthanol precipitation of plasmid DNA
B
Resuspend plasmid DNA in Tris-CDTA
or sterile nanopure water
1.5 ml microcentrifuge tube
Obtain pure form I plasmid DNA by
acid-phenol extraction
Fig. 1. Schematic outline of the alkaline $&/acid-phenol plasmid purification procedure.
6 Aubin, Weinfeld, and Paterson
2.1. lFiton &ysislCsCl Double Banding Method
1. Superbroth: Superbroth is prepared from stock solutions A and B.
To prepare solution A, mix 120 g of tryptone, 240 g of yeast extract,
and 50 mL glycerol in 9000 mL of water. Prepare solution B by
mixing 125 g of %HPO, and 38 g of KHgO,in a total vol of 1 L.
Sterilize each solution separately in the autoclave. Prepare 1 L of
Superbroth by combining 900 mL of solution A and 100 mL of
solution B. The final pH should be 7.2.
2. TE: 10 mMTris-HCl, pH ‘7.9; 1 mMEDTA, pH 8.0.
3. TES: 50 mM Tris-HCl, pH ‘7.5; 40 mM EDTA; 25% (w/v) sucrose.
Sterilize by filtration (0.22 w).
4. 0.25MEDTA, pH 8.0.
5. Triton solution: Mix together 1 mL of 10% Triton X-100; 31.5 mL 0.25M
EDTA, pH 8.0; 5 mL of 1 M Tris-HCl, pH ‘7.9; and 62.5 mL of water.
Sterilize the solution by filtration (0.22 pm) and store at 4°C.
6. Ethidium bromide: 10 mg/mL in 10 mMTrisHC1, pH 7.5. This solu-
tion does not require sterilization.
‘7. 5MNaCl.
8. HPLCgrade ethanol (95%).
9. Isopropanol.
2.2. Large-Scale Isolation
of Plasmid DNA by Alkaline Lysis
1. LB (Luria-Bertani) broth: Dissolve 10 g of tryptone, 5 g of yeast extract,
and 10 g of NaCl in 900 mL ofwater and adjust the pH to 7.5. Complete
to 1 L with water and sterilize immediately in the autoclave.
2. NTE buffer: 10 mMTris-HCl, 100 mMNaC1, 1 mMEDTA (pH ‘7.5).
3. PEB I: 50 mM n-glucose, 25 mM Tris-HCl, 10 mM CDTA (pH 8.0).
Sterilize the solution by filtration (0.22 pm) and store at 4OC. Do not
autoclave. PEB I is stable indefinitely if stored under aseptic conditions.
4. PEB II: 0.2N NaOH, 1 .O% SDS. Clarify the solution by filtration (0.22 p) .
Do not autoclave. Store at room temperature. PEB II is stable for at least 3 mo.
5. PEB III: 3Mpotassium acetate, 1.8Mformic acid. Clarify the solution by
filtration (0.22 pm) and store at room temperature. Do not autoclave.
PEB III is stable for at least 3 mo.
6. Acetate-MOPS: 100 mMsodium acetate; 50 mMMOPS, pH 8.0. Clarify
the solution by filtration (0.22 pm) before sterilizing in the autoclave.
7. LiCL-MOPS: 5MLiCl; 50 mMMOPS, pH 8.0. This solution tends to be
turbid even if high-purity LiCl is used. Clarify the solution by filtration
(0.45 pm) before sterilizing in the autoclave.
Plasmid DNA Preparation 7
8. RNAse buffer: 50 mMTris-HCl, 10 mMNaC1, 10 mMEDTA (pH 7.5).
9. RNAse A (DNAse-free): Dissolve lyophylized RNase A to a final concen-
tration of 1 mg/mL in sterile 5 mM Tris-HCl, pH ‘7.5. Hold the
preparation at 80°C for 15 min to inactivate traces of contaminating
deoxyribonucleases, and allow to cool at room temperature for 20 min.
Dispense 1 00-PL vol in sterile microcentrifuge tubes and store at -2OOC.
Individual tubes may be thawed and refrozen repeatedly without signifi-
cant loss of enzyme activity.
10. RNAse Tl : Concentrated RNase Tl (125,000 U/mL) is diluted to a
final concentration of 500 U/mL in sterile 5 mMTris-HCl, pH ‘7.5, and
stored at 4OC.
11. 10% SDS: Dissolve 10 g of molecular biology grade sodium dodecyl
sulfate (SDS) in 100 mL of autoclaved nanopure water. Clarify the so-
lution by filtration (0.22 pm) and store at room temperature. Do not
autoclave.
12. Proteinase K: Dissolve lyophylized proteinase K to a final concentration
of 20 mg/mL in autoclaved nanopure water. Dispense lOO-pL vol in
sterile microcentrifuge tubes and store at -2OOC. Individual tubes may
be thawed and refrozen repeatedly without significant loss of potency.
13. T&-buffered phenol (pH 8.0): The use ofhigh-purity (redistilled) phenol
is critical. Begin by melting crystallized phenol in a 65OC water bath.
Add an equalvol of lMTris-HCl, pH 8.0/0.2% ~mercaptoethanol. Shake
vigorously to create an emulsion, and allow the phases to separate by
gravity. Remove the upper aqueous layer by aspiration and equilibrate
the phenol twice with an equal vol of O.lM Tris-HCl, pH 8.0/0.2%
pmercaptoethanol. Store the buffered phenol in the dark at 4OC under
a film of O.lMTris-HCI, pH 8,0/0.2% pmercaptoethanol. The prepa-
ration remains stable for at least 3 mo under these conditions.
CAUTION: Phenol is a strong oxidant and can cause severe burns.
Always wear gloves and exercise due care in handling. Phenol is also
light-sensitive. Always prepare and store in an amber glass bottle.
14. Phenol:chloroform (1:l): Add 1 vol of Tris-buffered phenol to an equal
vol of chloroform:isoamyl alcohol (24:l) (see below) in an amber glass
bottle. Store in the dark at 4OC under a film of O.lMTrisHCl, pH 8.0/
0.2% ~mercaptoethanol.
15. Chloroform:isoamyl alcohol (24:l): Mix 240 mL of chloroform and 10
mL of isoamyl alcohol in a glass amber bottle. Store in the dark at 4OC.
16. 2.5Msodium acetate, pH 5.5.
17. Tris-CDTA: 10 mMTris-HCl; 1 mMCDTA, pH 8.0.
18. HPLC-grade ethanol (95%).
8 Aubin, Weinfeld, and Paterson
19, Lyzozyme (lyophylized) .
20. Chloramphenicol: Prepare a 34mg/mL stock solution using 95% eth-
anol just prior to use.
21. Spectinomycin: Spectinomycin is usually present at 66% potency.
Prepare as required at a stock concentration of 30 mg/mL in nanopure
water. Sterilize by filtration (0.22 pm) before use.
2.3. Purification of Form I Plasmid DNA
by Acid-Phenol Extraction
1. 50 mMsodium acetate/75 mMNaC1, pH 4.0: To 400 mL of autoclaved
nanopure water, add 10 mL of 2.5Msodium acetate, pH 5.5, and ‘7.5 mL
of a 5MNaCl stock solution. Adjust the final pH exactly to 4.0 with gla-
cial acetic acid, and complete the vol to 500 mL. Sterilize the solution by
filtration (0.22 pm) 0nZy and store at room temperature.
2. 50 mM sodium acetate, pH 4.0: To 400 mL of autoclaved nanopure
water, add 10 mL of 2.5M sodium acetate, pH 5.5. Adjust the final pH to
4.0 with glacial acetic acid and complete the vol to 500 mL. Sterilize the
solution by filtration (0.22 pm) only and store at room temperature.
3. Acidified phenol: Melt high-purity (redistilled) phenol in a 65OC water
bath. Add an equal vol of 50 mM sodium acetate, pH 4.0, and shake
vigorously to create an emulsion. Allow the phases to separate by gravity
and remove the upper aqueous layer by aspiration. Repeat this proce-
dure twice. Store acidified phenol in the dark at 4OC under a film of 50
mM sodium acetate (pH 4.0). This product has a short shelf-life and
should be prepared either as required or on a biweekly basis.
3. Methods
3.1. Isolation of Form I Plasmid DNA
by lkiton Llysis Followed by CsCl Banding
1. Initiate a 5-mL culture from a single colony (or frozen stock) of bacteria
containing the plasmid at 37°C overnight. Use the entire culture to seed
800 mL of Superbroth, and incubate for 36 h at 37°C with vigorous
shaking.
2. Collect the bacteria by centrifigation at 5000gfor 10 min (4OC), using a
Sorvall GS-3 rotor (or its equivalent; DuPont).
3, Resuspend the bacteria in 100 mL of TE. Centrifuge again to pellet the
material. The sample may be stored frozen (-20°C) at this stage.
4. Resuspend the pellet thoroughly in 9 mL of ice-cold TES.
5. Add 0.9 mL of 10 mg/mL lysozyme (freshly prepared in TEYS) and hold on
ice for 5 min.
Plasmid DNA Preparation 9
6. Add 3.7 mL of 0,25MEDTA, pH 8.0, mix thoroughly, and hold on ice
for another 5 min.
‘7. Add 14.5 mL of ice-cold Triton lysis solution, mix thoroughly, and
hold on ice for 10 min.
8. Remove the debris by centrifugation (25,000 rpm in a Beckman SW2’7
rotor or its equivalent) for 30 min at 4OC.
9. Decant the supernatant into a 50-mL polypropylene tube and adjust the
weight of the liquid to 30.17 g with TE. Add 28.14 g of solid CsCl and
mix thoroughly until completely dissolved. Add 4.5 mL of ethidium
bromide solution and mix by inversion. CAUTION: Ethidium bromide
is a very strong mutagen. Always wear gloves while handling it.
10. Transfer the mixture to a Beckman polyallomer Quick Seal@ ultracen-
trifuge tube, and seal the vessel according to the supplier’s instructions.
A single preparation can be accommodated by the Beckman VTi 50 or
Ti 60 rotor. The VTi 50 rotor should be spun for at least 18 h at 45,000
rpm (20°C)) whereas the Ti 60 unit will require a run time of at least 60
h at 35,000 rpm (2OOC). NOTE: The new generation of Beckman rotors
and benchtop ultracentrifuges allows for smaller plasmid preparations
to be banded in much shorter time periods.
11. Following centrifugation, view the tubes under medium-wave (302-nm)
W light. CAUTION: Ultraviolet light is both mutagenic and carcino-
genic. Wear adequate protection (i.e., use a W-protective face shield
[eye goggles will not protect the rest of your face or neck], ensure that
your lab-coat sleeves cover your forearms, and wear latex gloves), Se-
cure the tube and insert a 19-gage syringe needle at the top to create an
air inlet. Puncture the side of the tube at a point just below the lower-
most band (form I plasmid; the upperband consists of open circular and
linear molecules) using a 20-mL hypodermic syringe equipped with a
19gage needle. Collect the banded material in a total vol of 4-5 mL.
12. Either reband the plasmid sample (seestep 13) or remove the ethidium
bromide with isopropanol saturated with CsCl solution at the concentra-
tion used for banding. Dialyse extensively against a large vol of TE to
remove the CsCl. Add l/10 vol of 5MNaCl and 2 vol of ethanol. Allow
the plasmid to precipitate overnight at -20°C, and collect the mate-
rial by centrifugation at 10,OOOg for 15 min at 4OC. Drain away the
ethanol carefully and air-dry the pellet to remove all traces of ethanol.
13. It is often necessary to reband the plasmid preparation. To do this,
prepare a 1.08 g/mL CsCl solution in TE. Add 0.1’7 mL of ethidium
bromide solution for each mL of TE used. Add the DNA sample obtained
in step 12 to the solution, mix thoroughly, centrifuge, and process as in
steps 1 O-21.
10 Aubin, Weinfeld, and Paterson
3.2. Large-Scale Isolation
of Plasmid DNA by Alkaline L&J
1. Revive a stock of plasmid-bearing bacteria in 5 mL of LB broth contain-
ing the antibiotic(s) required for plasmid selection. Incubate overnight
at 37°C with shaking. The next morning, transfer 2.5 mL of the satu-
rated culture to two 2-L Erlenmeyer flasks (each containing 500 mL of
sterile antibiotic/LB broth), and grow the bacteria at 37°C with vigorous
shaking (250 x-pm) until the OD, reaches 0.8. Add 2.5 mL of the 34
mg/mL chloramphenicol stock (final concentration, 170 pg/mL) to each
flask and allow plasmid amplification to proceed over 20 h at 37°C with
shaking. Plasmids containing a transcriptionally active chlorampheni-
co1 acetyl transferase cassette can be amplified using 150 pg/mL
spectinomycin.
2. Harvest the bacteria by centrifugation at 500gfor 8 min (4OC) in 500-mL
Oak Ridge bottles using a Sorvall GS3 rotor or its equivalent. Discard
the supernatant into a receptacle containing germicidal detergent or
liquid bleach.
3. Resuspend each bacterial pellet in 50 mL of ice-cold NTE buffer. Pool
the material in a single 250-mL Oak Ridge bottle and recover the washed
cells by centrifugation at 5000gfor 8 min at 4°C using a Sorvall GSA r-o-
tor or its equivalent. Discard the supernatant as in step 2.
4. Using a sterile l-mL plastic pipet as a stirring rod, gently dissolve the
pellet in 1 mL of icecold PEB I buffer. A homogeneous slurry should be
obtained. Add 9 mL of icecold PEB I buffer supplemented with 10 mg
of lysozyme, and mix thoroughly with the plastic pipet. Allow cell lysis to
proceed for 30 min in ice water (seeNote 2).
5. Add 20 mL of room-temperature PEB II and stir the mixture thoroughly
but gently with the plastic pipet. The lysate should become very viscous
and translucent. Hold in ice water for 15 min and stir occasionally.
6. Add 15 mL of room-temperature PEB III and stir the mixture vigorously
with the plastic pipet for several min until a coarse white precipitate
forms and the viscosity disappears. Hold in ice water for 30 min and
collect the debris by centrifugation at 10,OOOgfor 10 min (4OC).
7. Transfer the supernatant to a new 250-mL Oak Ridge bottle and add 2
vol (90 mL) of chilled (-20°C) ethanol. Mix well and precipitate the
nucleic acids at -70°C for 30 min. Collect the material by centrifugation
at 10,OOOg for 10 min (4OC).
8. Using a sterile pipet, gently dissolve the pellet in 5.0-7.5 mL of acetate-MOPS
buffer. Transfer the solution to a sterile 30-n& Oak Ridge or Corex@ tube and
precipitate the nucleic acids at -70°C for 30 min. Collect the material by
Plasmid DNA Preparation 11
centrifugation at 10,OOOg for 10 min (4OC) using a Sorvall HB4 or
SS34 rotor.
9. Decant the ethanol carefully and resuspend the pellet in W-7.5 mL of
autoclaved nanopure water. Measure the vol of the sample and add an
equal vul of LiCl-MOPS buffer. Mix well and place in ice water for 15
min. Centrifuge at 10,OOOg for 10 min (4OC). This step will precipitate
the bulk of the coextracted RNA and leave the plasmid DNA (with re-
sidual low-mol-wt RNA species) in solution.
10. Using a sterile pipet, transfer the supernatant to a new Oak Ridge or
Corex@ tube. Hold at 60°C for 10 min, and collect any residual precipi-
tate by centrifugation as in step 9 (see Note 3).
11. Divide the supernatant between two 30-mL Corex@ tubes and add 2 vol
of chilled ethanol. Hold at -70°C for about 1 h and recover the nucleic
acids by centrifugation at 10,OOOgfor 10 min (4OC).
12. Resuspend each pellet in 5.0-7.5 mL of acetate-MOPS buffer and com-
bine the material in one of the Corex@ tubes. Precipitate the sample with
2 vol of chilled ethanol as outlined in step 8. Repeat the precipitation
once. Decant the ethanol carefully and air-dry the pellet for 10-15 min.
13. Dissolve the pellet in 5.0-7.5 mL of RNase buffer. Add 10 pL/mL RNase
A stock (final concentration 10 pg/mL) and 10 pL/mL RNase Tl stock
(final concentration 5 U/mL) . Incubate at 37°C for 90 min.
14. Add 50 pL/mL 10% SDS stock (final concentration 0.5%) and 2.5 pL/
mL proteinase K stock (final concentration 50 pg/mL). Incubate for
2 h at 37°C.
15. Extract the aqueous mixture as follows: once with Tris-buffered phenol,
twice with phenol:chloroform (l:l), and once with chloroform:isoamyl
alcohol (24:l). Disposable polypropylene culture tubes (sterile; 17 x
100 mm) are very practical for this purpose. For each extraction step,
add an equal vol of the organic solvent, create an emulsion by shaking
(do not use a mechanical vortex), and separate the phases by centrifu-
gation at 5000g for 5 min (25°C). A Sorvall HB4 rotor allows sharp
phase separation. Transfer the upper aqueous phase to a new tube fol-
lowing each extraction step.
16. To the final aqueous phase, add l/10 vol of 2.5Msodium acetate, pH 5.5,
and 2 vol of chilled ethanol. Precipitate the plasmid DNA for 20 min at
-70°C and centrifuge as described in step 8.
17. Clean the plasmid DNA twice by acetate-MOPS/ethanol precipitation as
described in step 12. Dry the DNA pellet briefly under vacuum and
dissolve in 1 mL of sterile Tris-CDTA buffer. Avoid using a mechanical
vortex to assist solubilization. Determine the concentration of the plas-
mid DNA stock by taking the absorbance of a l/20 dilution at 260 nm
12 A&in, Weinfeld, and Paterson
and assuming that 1 .O OD, unit is equivalent to a concentration of 50
pg DNA/mL. For example, mix 25 /..tLo f the DNA preparation in 475
PL of Tris-CDTA. Using a 0.5 mL quartz cuvet (glass cuvets absorb
sign&an tly in the W spectrum), “zero” the spectrophotometer using
Tris-CDTA. Take the OD reading of the DNA and calculate its concen-
tration as follows:
(reading at 260 nm) x 20 (l) X 50 W= [DNA] in pg/mL
where (I) is the dilution factor and (*) is the conversion factor.
The sample should also be scannedwithin the 200-to 300-nm range in order
to obtain a rough measure of the purity of the preparation. The scan should
show two peaks, the first around 205 nm (attributed to the Tris-CDTA) and
the other at 260 nm (contributed by DNA), A high-purity DNA preparation
should show no evidence of a shoulder beyond 260 nm, and the 260 nm/280
nm ratio should be >1.8.
If care is taken throughout the procedure to pipet gently and mini-
mize the use of a mechanical vortex, form I plasmid DNA is routinely
obtained at >90% yield for plasmids of 15 kbp or less, as verified by aga-
rose gel electrophoresis.
3.3. Purification of Form I Plasmid DNA
by Acidified-Phenol Extraction
1, Transfer the DNA stock to a sterile 1.5-mL microcentrifuge tube. Add
l/10 vol2.5Msodium acetate, pH 5.5, mix well, and add 2 vol of chilled
ethanol. Precipitate the DNA at -‘70°C for 30 min and recover by cen-
trifugation at 10,OOOg for 10 min (4OC). Air-dry the pellet for 15 min at
room temperature.
2. Gently redissolve the sample to a final concentration not exceeding250
yg/mL in 50 mM sodium acetate/‘75 mMNaC1, pH 4.0. A total vol
of 400-500 I,~L is practical (see Notes 4 and 5).
3. Add an equal vol of acidified phenol and shake vigorously by hand
(DO NOT VORTEX) for 2-3 min. Separate the phases by centrifuga-
tion at 10,OOOgfor 60 s at room temperature.
4. Transfer the upper aqueous layer to a sterile 1.5-mL microcentrifuge
tube and precipitate the plasmid DNA as outlined in Step 1. Redissolve
the pellet in 250 PL of acetate-MOPS buffer and precipitate the DNA
with 2 vol of chilled ethanol. Repeat this step once. Resuspend the DNA
sample in 0.5 mL of TrisCDTA. Determine the concentration and verify
the integrity of the preparation by spectrophotometry and agarose gel
electrophoresis, respectively. One round of acid-phenol extraction is
generally sufficient to achieve a high degree (i.e., > 98% form I) of plas-
mid purification.