MEGA- and GIGA-FILTER preps of cosmid-, BAC-,
PAC, YAC-, and P1-DNA with JETSTAR 2.0
(September 2005)
Introduction + General Considerations!
Cosmids as well as BAC-, PAC-, YAC- or P1 constructs can be purified on the MEGA and
GIGA scale with JETSTAR 2.0 using the standard plasmid protocol with some modifications.
The above-mentioned DNA species behave like very low copy plasmids (in many cases
present with even only 1 copy per cell). This makes it necessary to process high amounts of
E. coli culture (2.5 - 5 litres) to obtain reasonable DNA yields. Therefore this protocol is
outlined for the maximum amount of E. coli culture.
Dealing with high volumes of bacterial culture makes it necessary to increase the volumes of
buffers E1 - E3 used for the preparation of the cleared lysate. A second aspect that must be
mentioned in connection with high amounts of bacterial culture is the potential risk of
residual RNA in the eluate. This protocol gives hints and suggestions for this aspect.
Another factor is the large size of these constructs, making it useful to use prewarmed
buffer E6 for elution.
Most protocols are recommending LB broth or a very similar variant plus appropriate
antibiotic as the medium of choice for the propagation of E. coli cells containing DNA
constructs.
While LB and its variants is good enough to let E. coli grow to high cell densities, it is not the
optimal choice for obtaining high DNA yields. With GENOMED's JETSTAR 2.0 you can use
all specialized media that are offered for high DNA yields (e.g. PGF series from
Qbiogene/MP Biomedicals, ‚Magnificent Broth‘ from McConnell Research, Terrific Broth, 2 x
YT).
The handling of the novel JETSTAR 2.0 MEGA/GIGA FILTER system is quite different
from the Mini/Midi/Maxi kits, that are based on gravity-flow columns. Therefore, new
users are strongly advised to read the entire protocol very carefully before starting
the procedure!
• Before using the kit for the first time, dissolve the lyophilized RNase (provided with the kit)
completely in 1.0 – 1.5 ml of buffer E1. Transfer this concentrated solution back into the
buffer E1 bottle and store the reconstituted buffer at 4°C. The final RNase concentration
is 100 µg/ml. The RNase-containing buffer E1 is stable for 6 months when stored at 4°C.
• Check buffer E2 for SDS precipitation. If SDS is precipitated in buffer E2 due to low
transport or storing temperatures, redissolve it by warming in a water bath at 37°C for a
few minutes.
A new working principle!
Although still being based on anion-exchange chromatography, the JETSTAR 2.0
MEGA/GIGA FILTER system is different from the hitherto known Mini/Midi/Maxi columns.
The MEGA/GIGA FILTER system is not based on gravity flow-columns, but consists of the
JETSTAR 2.0 anion-exchange resin packed in a vacuum-driven filter cartridge.
Additionally, the new FILTER system contains an extra filter cartridge for clarification of the
bacterial lysate. Vacuum (–600 to –800 mbar or -20 inch Hg) is applied to the cartridge by a
conventional water jet filter pump (being standard equipment in nearly all molecular biology
labs) or a membrane vacuum pump.
What auxiliaries do you need?
The JETSTAR 2.0 MEGA/GIGA FILTER system requires the presence of:
• A water jet filter pump (or equivalent vacuum source), capable of generating a negative
pressure of –600 to –800 mbar (approx. -20 inch Hg),
• a 500 ml or 1 litre vacuum-resistant laboratory bottle with 45 mm neck (i.e. DURAN ) for
collection of the cleared lysate,
• a 1 litre vacuum-resistant laboratory bottle with 45 mm neck (i.e. DURAN ) for
equilibration as well as binding and washing steps,
• a 100 ml vacuum-resistant laboratory bottle with 45 mm neck (i.e. DURAN ) for the
elution step.
To avoid the possibility of implosion, do not use plastic/glass bottles or any other
vessels that are not designed for the use with vacuum. Do not use plastic/glass
bottles or any other vessels that are cracked or scratched. Wear safety glasses when
working near a bottle under vacuum.
Cartridges for lysate filtration and DNA preparation
Cartridges for filter clarification of the bacterial lysate are containing a yellow ring.
Cartridges with ion-exchange chromatography material for the purification of DNA are
containing a blue ring.
Protocol:
1.)
Inoculate a bacterial culture containing your DNA-construct of choice in a suitable
medium (for volumes see step 2) with the appropriate antibiotic and grow the bacteria
for 16 - 24 h (usually overnight).
2.)
Decide, what cartridge type (MEGA/GIGA) should be used. For the different prep-sizes
use the following amounts of bacterial culture:
-MEGA:
up to 2.5 litres,
-GIGA:
up to 5 litres.
3.)
Setup of FILTER cartridge: For lysate filtration check for the cartridges with the
YELLOW ring. Screw the MEGA FILTER cartridge onto a clean 500 ml bottle or the
GIGA FILTER cartridge onto a clean 1 litre bottle. Each bottle must have a 45 mm
neck fitting to the cartridges.
Note: Do not overtighten the FILTER cartridges on the bottle neck, since the plastic
may crack.
4.)
Harvesting bacterial cells: E. coli cells are pelleted by centrifugation for 3 min at
13,000 x g. Remove all traces of medium carefully.
5.)
Cell Resuspension: Add 100 ml (MEGA prep) or 250 ml (GIGA prep) of
buffer E1 to the pellet and resuspend the cells until the suspension is
homogeneous. No cell clumps must be visible.
Normally 100 µg/ml RNase should be enough to get rid of all RNA. If residual RNA
tends to be a problem, increase the amount of RNAse in buffer E1 to 400 µg/ml. In
addition to that, buffer E1 can be supplemented with 100 U/ml of RNase T1. The
combined activities of RNase A and T1 will result in a better digestion efficiency of the
bacterial RNA, thus leading to a better removal of the RNA during the column
procedure.
6.)
Cell Lysis: Lyse the bacterial cells by adding 100
ml (MEGA prep) or 250 ml
(GIGA prep) of buffer E2. Mix gently but thoroughly until a homogeneous
lysate is obtained. Due to the release of genomic DNA the mixture is very viscous at
this stage. DO NOT VORTEX as this will result in shearing of the genomic DNA!
Incubate at room temperature for 5 min.
7.)
Neutralization: Neutralize the lysis mix from step 6 with 100 ml (MEGA prep) or
250 ml (GIGA prep) of buffer E3. Mix gently but thoroughly until a
homogeneous mixture is obtained. DO NOT VORTEX! The liquid of the neutralized
lysate must be completely thin-bodied again (not longer viscous). NO remainders of
the viscous lysate must be left!!!
A white, flocculent precipitate made of proteins, cellular debris, genomic DNA and
detergent will form.
8.)
Filtration: After mixing with buffer E3 pour the as much bacterial lysate from step 7 as
possible into the prepared MEGA or GIGA FILTER cartridge from step 3. Let stand at
room temperature for at least 10 min without agitation!! Then attach a vacuum
source to the tubing connector and apply vaccum. Collect the clear flowthrough into
the bottle. During the filtration process pour the remainder of the lysate into the
cartridge. Keep the vacuum on until all liquid has drained from the unit. Then switch off
the vacuum source.
Important Note: It is very important to let the lysate stand for at least 10 minutes after
the transfer into the cartridge. This is for the precipitate to float and form a layer on top
of the lysate. For a quick and convenient filtration it is vital, that the majority of
precipitate forms the layer on top of the liquid. If necessary, extend the incubation time
of the lysate in the cartridge until the layer has formed. This ensures convenient
filtration without clogging.
9.)
Add 100
mls of buffer E5 to the MEGA or GIGA FILTER cartridge and gently
stir the precipitate with a sterile spatula. Connect the vacuum source again and apply
vacuum until all liquid has been pulled through completely. Close the bottle and mix the
contents by shaking. The bottle now contains the filtered lysate with plasmid DNA.
Note: Gentle agitation of the precipitate improves the flow of liquid through the filter
unit.
10.) Equilibration: For DNA preparation check for the cartridges with the BLUE ring.
Screw the MEGA or GIGA cartridge onto a 1 litre laboratory bottle with 45 mm neck
(i.e. DURAN ) and fill in 100 ml (MEGA prep) or 200 ml (GIGA prep) of
equilibration buffer E4. Apply vacuum to the cartridge through the side-arm
with tubing-connector and suck through the complete amount of liquid. Keep the
vacuum on until all liquid has drained from the resin. Discard the flowthrough.
11.) Loading of the lysate: Fill the cleared lysate from step 9 into the cartridge with the
equilibrated JETSTAR 2.0 resin and apply vacuum to the cartridge through the sidearm with tubing-connector. Keep the vacuum on until all of the lysate has passed
through the resin. You may take an aliquot of the flowthrough for further analysis.
12.) Wash 1: Fill 150
ml (MEGA prep) or 275 ml (GIGA prep) of modified
wash buffer ‚E5-850 (pH 4.0)‘, that contains 850 mM NaCl instead of 800
mM and has a pH of 4.0 (recipe see below) into the cartridge and apply vacuum to
the cartridge through the side-arm with tubing-connector. Keep the vacuum on until all
liquid has drained from the resin. You may take an aliquot of the flowthrough for further
analysis.
13.) Wash 2: Repeat step 12 once with 150
ml (MEGA prep) or 275 ml (GIGA
prep) of modified wash buffer ‚E5-850 (pH 4.0)‘. Two successive
rounds of washing should be sufficient to remove all impurities (i.e. proteins, degraded
RNA, metabolites, dyes) completely.
14.) Plasmid Elution: Take off the filter cartridge from the 1 litre bottle and screw it onto a
clean, sterile 100 ml laboratory bottle with 45 mm neck (i.e. DURAN ).
Apply 50 ml (MEGA prep) or 100 ml (GIGA prep) of prewarmed
(55-60°C) elution buffer E6 into the cartridge.
IMPORTANT NOTE: Apply a soft vacuum (–100 to –200 mbar) to the cartridge
through the side-arm with tubing-connector until approximately 10 – 20 ml
(MEGA prep) or 20 – 30 ml (GIGA prep) of buffer E6 have eluted from the
cartridge. Then release vacuum from the cartridge, so that no further liquid is
pulled through the resin. Let stand for 1 min without agitation. Then switch on
vacuum again and draw the remaining liquid from the resin into the receiver
bottle. Keep vacuum on for approximately 5 min until all liquid has drained from
the resin. You may take an aliquot of the eluate for further analysis.
The final DNA yield can be increased by approximately 10-15% if a second elution step
with another 50 ml (MEGA prep) or 100 ml (GIGA prep) of buffer E6 is carried out as
described.
15.) Plasmid Precipitation: Precipitate the DNA with 0.7 volumes of isopropanol. Spin
down the DNA for at least 30 min at ≥13,000 x g and 4°C. Discard the supernatant,
wash the precipitated DNA with 20 ml of 70-80% ethanol per tube and re-centrifuge for
5 min as described.
Discard the ethanol and air-dry the DNA pellet completely until all ethanol has
evaporated. Be careful when using vacuum to not overdry the DNA. If this occurs,
the DNA will be very recalcitrant to redissolving. A good alternative to vacuum is drying
at elevated temperatures, i.e. for 10 min at 50-55°C in a water bath or thermo block.
Redissolve the DNA in a suitable volume of buffer. Suitable solutes are i.e. 10 mM
Tris-HCl (pH 7.0 – 8.5), TE buffer or simply water.
Plasmid DNA is quite sticky and tends to spread over the whole wall of the centrifuge
tube if a fixed angle rotor is used. Therefore we recommend the use of a swing-out
rotor that allows centrifugal forces of ≥12,000 x g (i.e. HB-4 or HB-6 for Sorvall
centrifuges). If such a rotor is not available, we recommend siliconization of the
centrifuge tubes with a repellent silane (i.e. dimethyldichlorosilane).
Recipe for modified buffer E5
The recipe for 1 litre of modified buffer E5 with 850 mM sodium chloride and a pH of 4.0
is as follows:
Final concentration
Substance
Amount
(grams)
100 mM sodium acetate (pH 4.0)
Sodium acetate
8.203 g
Glacial acetic acid (99%)
15,00 g
850 mM NaCl
Sodium chloride
49,67 g
Final volume:
Adjust with water to:
1 liter
All ingredients, which means solids and liquids are weighed in gravimetrically! The
final pH value may differ slightly (± 0.1) depending on the respective pH meter used. Such
slight deviations can be tolerated. In no case the pH value should be re-adjusted.
All chemicals should be of the highest quality and purity (at least 'p.a.' grade). The water
should be either double-distilled or of Milli-Q (Millipore) quality.
The acetic acid used for pH adjustment should be taken from a fresh bottle, that was
always tightly closed when not in use.
JETSTAR 2.0 Buffer Compositions:
Buffer E1: 50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 100 µg/ml RNase A
Buffer E2: 200 mM NaOH, 1%(w/v) SDS
Buffer E3: 3.1 M potassium acetate (pH 5.5 with acetic acid)
Buffer E4: 100 mM sodium acetate (pH 5.0 with acetic acid), 600 mM NaCl, 0.15% Triton X-100
Buffer E5 (standard): 100 mM sodium acetate (pH 5.0 with acetic acid), 800 mM NaCl
Buffer E6: 100 mM sodium acetate (pH 5.0 with acetic acid), 1.500 mM NaCl