Caltech Genome Research Laboratory
California Institute of Technology
The Crew:
Dr. Ung-Jin Kim, Director
Phone: (818)395-4901 (office) 4154 (laboratory)
Fax: (818)796-7066
Address:
Division of Biology, 147-75
California Institute of Technology
Pasadena, CA 91125,
U.S.A.
ung@ash.tree.caltech.edu
Dr. Hiroaki Shizuya, Director
Phone: (818)395-4154
Fax: (818)796-7066
Address:
Division of Biology, 147-75
California Institute of Technology
Pasadena, CA 91125,
U.S.A.
shizuya@cco.caltech.edu
Dr. Melvin I. Simon, Professor
Phone: (818)395-3944
Fax: (818)796-7066
Address:
Division of Biology, 147-75
California Institute of Technology
Pasadena, CA 91125,
U.S.A.
simonm@starbase1.caltech.edu
Yu-Ling Sheng, Staff
Valeria Mancino, Staff
Kevin Roche, Staff
Tatiana Slepak, Staff
Yu-Jiun Chen, Staff
Cecilie Boysen, Visiting Student
Hyung Lyun Kang,Visiting Student
Sun Shim Choi, Visiting Student
The Projects:
Human Genome Project
Construction of Human Bacterial Artificial Chromosome (BAC) Library resource:
supported by the U.S. Department of Energy
Physical mapping of Human Chromosome 22 using BAC clones and YAC frameworks:
supported by the U.S. Department of Energy
in collaboration with Drs. Ian Dunham (e-mail:id1@sanger.ac.uk), Charmain L. Garrett,
and Luc J. Smink at The Sanger Center
Mouse Genome Project
Construction of Mouse Bacterial Artificial Chromosome (BAC) Library resource:
supported by the U.S. Department of Energy
in collaboration with Dr. Bruce Birren at Whitehead Institute, MIT (e-mail:bwb@genome.wi.mit.edu)
Dr. Birren was formerly a member of Caltech Genome Group
Microbial Genome Project
Sequencing the 1.8 Megabase Genome of the Archaeum Pyrobaculum aerophilum
supported by the U.S. Department of Energy
in collaboration with Dr. Jeffrey Miller and Sorel Fitz-Gibbon (e-mail:sorel@ewald.mbi.ucla.edu)
References: Some selected literatures from our work
How to build a BAC library:
The vectors
The most important aspect of our cloning vectors is that they are based on
the E. coli F-factor replicon. It allow for strict copy number control of the
clones so that they are stably maintained at 1-2 copies per cell. The stability
of the cloned DNA during propagation in E. coli increases dramatically in lower
copy number vectors (Kim et al, NAR, 20(5):1083-1085). The stabilization is
especially remarkable for the genomic DNA that are normally unstable in E. coli.
They include genomes of certain Archaea, or DNA of mammalian or other origins
that contain numerous repetitive sequences.
For example, stable Fosmid libraries have been generated from the genomes of an
Archaeum and the Sea urchin that were highly unstable/unclonable in multicopy
cosmid vectors.
The pBeloBAC11 vector
This vector allows lacZ-based positive color selection of the BAC
clones that have insert DNA in the cloning sites at the time of
library construction. Because the vector exist in single copy in
E. coli, purifying the DNA in large quantity takes some effort.
Therefore, we have been supplying the vector as an E. coli strain
that carries the vector. Please see the experimental protocol
below to find out how to prepare large amounts of pure pBeloBAC
DNA.
The pBAC108L vector
The very first version of BAC vector. After transformation, clones
carrying human DNA insert had to be selected by colony hybridization
with labeled human DNA.
The pFOS1 vector
This single copy cosmid vector was constructed by fusing pBAC108L and
pUCcos (a pUC vector in which the region including lacZ and multiple
cloning sites) was replaced by lambda cos sequence. In vivo homologous
recombination between two vectors via cos sites resulted in pFOS1.
The vector is extremely unstable in most of E. coli strains due to the
presence of double cos sites. pop2136 strain (Methods in Enzymology
vol.152 pp173-180, 1987), for no apparent reason, can maintain pFOS1
(and other double-cos cosmid vectors) with some stability. The bireplicon
is driven by the pUC replication origin, and exists in high copies in
E. coli. After in vitro packaging and transfection to E. coli, Fosmids
are exactly the same as pBAC108L clones and exist as miniBACs with 40
kb inserts. Fosmid library can easily be constructed using the protocol
for constructing cosmid libraries with double-cos vectors. The Fosmid
system is useful for quickly generating miniBAC libraries from small
amounts of DNA, such as flow-sorted chromosomal DNA.
A large quantity of the vector has been prepared by CsCl-prep, and has
always been available from us for anyone interested in constructing
one's own Fosmid library.
Inquiries should be forwarded to: Dr. Ung-Jin Kim
Introduction
Bacterial Artificial Chromosomes (BACs) were developed in our group to clone
and stably maintain large DNA fragments.
The BAC and Fosmid vectors have been derived from a mini-F plasmid
(O'Connor et al. 1989) and contains the minimal sequences needed for the
autonomous replication, copy number control, and partitioning of the plasmid.
The genes labeled oriS, repE, parA, parB, and parC are derived from the F factor
and encode proteins required for these functions. The cloning site in pBeloBAC
was obtained from pGEM3 (Promega Biochemical) and contains all the restriction
sites from the multiple cloning site of that vector. However, only the HindIII,
BamHI, and SphI sites are unique. The cosN site from bacteriophage lambda can
be cleaved with the commercially available enzyme lambda terminase to yield a
single linear molecule. The two cohesive ends produced by terminase are
non-identical, and therefore may be specifically labeled with oligonucleotides
directed at either of the two ends. The cosN can be used for restriction mapping
of the cloned DNA.
The loxP site is from the bacteriophage P1, and can be used for P1 Cre protein
mediated site specific recombination, which will allow for linearization and
other manipulation of the BAC or Fosmid clones.
PREPARATION OF BAC VECTOR
A. Purification of Vector
Because the BAC vectors exists as single copy plasmids, large volumes of cultures
are necessary to obtain microgram quantities of pure DNA. A few liters of
culture in LB medium will yield decent amounts of the vector DNA, usually less than
20 micrograms. However, an entire human or mouse library can be constructed from
less than a microgram of the vector DNA. E. coli DNA contamination will hamper
library construction, and should be avoided. Therefore, the purity of the vector
DNA is much more emphasized than the yield.
1. Prepare three 3-liter wide-mouth culture flasks, each containing
1 liter of LB Broth with 12.5µg/ml of chloramphenicol. Inoculate
with 1 ml of a freshly grown, saturated overnight culture of DH10B
(Grant et al. 1990) containing the BAC vector, prepared from a single
freshly streaked blue colony from an X-gal/IPTG plate.
2. Grow to late log phase at room temperature with shaking at 200 rpm.
This will take approximately 24 hours if starting with a 1:1000 dilution
of an overnight culture. Cultures which are held in stationary phase
will exhibit reduced DNA yields.
3. Harvest the cells by centrifugation for 10 minutes at 4,000 xg at 4û C.
4. Resuspend cell pellets by pipetting up and down in Solution I (without
lysozyme), using 25 ml for each liter of original bacterial culture.
When the cells are well resuspended, pool all bacteria and transfer
to a single centrifuge bottle. Add lysozyme to 2 .5 mg/ml, and mix by
inversion. Incubate 5' at room temperature.
5. Add 50 ml of Solution II for each liter of original culture. Mix well
by inversion, leave on ice for 10'. As the cells lyse, the resulting
mixture will become extremely viscous.
6. Add 37 ml Solution III for each liter of original culture. Mix gently
by swirling until the suspension develops a flaky, curd like precipitate
without large chunks. Incubate on ice for 10'.
7. Centrifuge 30' at 8,000 xg (Sorvall GS3 rotor) at 4û C.
8. Promptly decant the supernatant and filter it by passing it through
a funnel lined with several layers of sterile cheesecloth. Measure
the volume and add DNase-free RNase to a final concentration of 0.1mg/ml.
Incubate at room temperature for 15-30 minutes.
9. During the RNase digestion, equilibrate 4 Qiagen-tip 500 columns with
10ml/column of buffer QBT (note the buffers QBT, QC, and QF as those
provided by Qiagen accompanying the columns).
10. Apply supernatant to the columns.
11. Wash each column twice with 2 x 30 ml of buffer QC.
12. Elute the DNA using 15 ml of buffer QF for each column.
13. Precipitate the DNA as follows: Adding 0.7 volumes of room temperature
isopropanol, mix by inversion and without further incubation centrifuge
at 15,000xg for 30' at 4' C.
14. Wash the DNA pellet with 15 ml of ice cold 70% ethanol, drain well and
air dry.
15. Resuspend DNA in 18.6 ml of TE. Add 20.5g CsCl and mix until the salt
is dissolved. (These volumes will allow the entire sample to be spun
in two tubes (16 x 76 mm) with the Beckman 70.1Ti rotor). For precise
control of the position of the bands in the centrifuge tube, measure
and adjust the refractive index of the DNA CsCl solution to 1.394 by
adding either TE or a saturated solution of CsCl in TE.
16. Add 0.4 ml of Ethidium Bromide (10mg/ml), mix, and fill centrifuge
tubes by "topping off" with a solution of CsCl prepared in TE (same
ratio of CsCl to TE as in step 15 above).
17. Balance and seal the tubes.
18. Centrifuge at 15¡ C in either a swinging bucket, fixed angle, or
near vertical angle centrifuge rotor. Centrifugation for between
55-70 hours at 45,000 rpm in a Beckman 70.1 Ti rotor produces the desired
separation.
19. Two bands should be clearly visible under a hand held long wave UV light,
and they should be well separated (>5mm apart). Remove the lower band
containing supercoiled vector DNA by puncturing the tube from the side
using a 1 cc syringe with an 18-gauge needle. Withdraw DNA slowly to
avoid any contamination of the sample with the upper band, which contains
chromosomal DNA and open circular vector. The volume withdrawn will
be approximately 0.5 ml from each 16 x 76 mm tube.
20. Remove the Ethidium Bromide by extracting 3-4 times with an equal
volume of isoamyl alcohol.
21. Dialyze the DNA for at least two hours against 2 liters of TE at 4û C.
22. Ethanol precipitate the DNA and rinse the pellet with 70% ethanol.
Drain well and air dry.
23. Redissolve the DNA in 50µl of TE. Digest a small aliquot (0.5 µl)
with HindIII or BamHI to linearize the vector and separate it on a
mini-gel using known amounts of size markers to determine vector
concentration and to verify the absence of contaminating chromosomal
DNA (which would be apparent as a broad smear in the gel).
24. Typical yields are about 2-4 µg plasmid/liter of culture.
25. DNA may be stored in TE frozen at -20û C for at least 6 months.
SolutionI: 25mM TrisHCl, pH 8.0; 50mM Glucose; 10mM EDTA
SolutionII: 0.2N NaOH; 1% SDS
SolutionIII: 5M Potassium Acetate, pH 4.8: Add glacial acetic acid to a solution of
3M potassium acetate to achieve a pH of 4.8. This is accomplished by adding
a minimal amount of water to the potassium acetate and then adding the acetic
acid until the potassium acetate is dissolved and the pH has reached 4.8.
B. HindIII Digestion
1. Digest approximately 2-3 µg of CsCl purified BeloBAC in a reaction volume of
100-200 µl. Use between 10 and 20 units of enzyme per microgram of DNA at
37û C for 1-2 hours. Use of the minimum amount of enzyme necessary to give
complete digestion will reduce the risk of degradation of the cloning site.
Degradation is evidnenced by the production of white colonies after self
ligation of digested vector. Promega sells restriction enzymes that have been
quality tested using self ligation and a blue/white color screen similar to
that used for BAC cloning.
2. Verify digestion by running 2-4 µl of digestion mix on a thin (<3 mm) minigel,
using lambda HindIII fragments as size markers.
3. To further test the extent of HindIII digestion, as well as to ensure that
the ends of the vector have not been harmed, set up small scale test ligations
using the HindIII cut vector. Set up parallel ligations containing 2-3 ng
of cut vector in a 10 µl reaction volume in the presence or absence of DNA
Ligase (New England Biolabs). For these small scale reactions we dilute
the ligase in 1X ligase buffer and use just 10 Units of enzyme. Ligate at
room temperature for 2 hours.
4. Dilute ligations 1:5 with water.
5. Electroporate 1 µl of each of the diluted reactions using 25 µl
electrocompetent cells (DH10B) with electroporation settings of 200 ohms,
2.5 kv and 25 microFarad.
Immediately after electroporation, add 1 ml of SOC (Appendix), transfer the
cells to glass culture tubes and grow for 45 min at 37 ûC with shaking at
200-300 rpm.
Spread small aliquots (0.05, 0.5, 5 and 50 µl of each) onto LB agar plates
(Appendix) containing 12.5µg/ml of chloramphenicol, 50µg/ml Xgal and
25 µg/ml IPTG.
6. Allow 18 hours of growth and color development and score the plates for the
number of both white and blue colonies. If the numbers are as expected,
proceed to dephosphorylation (see below for discussion of appropriate colony
numbers).
7. To dephosphorylate the linearized vector, combine the following: 2-3 mg of
HindIII cut vector in 100 ul 1X restriction digestion buffer, 11 ul 50 mM
CaCl2 (final conc. approx. 5 mM) and 20-30 units HK Phosphatase.
Incubate at 30û C for 1 hour. It is convenient to use a heat sensitive
phosphatase to avoid phenol extraction and the inevitable loss of sample.
HK Phosphatase TM from Epicentre Technologies requires only the addition of
calcium to restriction reactions to be fully active. If the vector has been
stored at 4 or -20û C, heat the sample at 65û C for 5 minutes, and cool it
to room temperature prior to adding phosphatase to release any sticky ends
which may have annealed. On occasion, more than the expected amount of
phosphatase is required for complete dephosphorylation.
It can be useful to set up several reactions using an amount of enzyme 5-fold
and 10-fold higher than the stated amount, to determine the level that will
be maximally effective.
8. Inactivate the enzyme by incubation at 65û C for 30 minutes.
9. Verify dephosphorylation through the use of test ligations. Set up the
following reactions in 10 ul volumes as described in step 3 above:
(i) dephosphorylated vector without ligase, (ii) dephosphorylated vector with
ligase, (iii) dephosphorylated vector with ligase and DNA serving as a test
insert. This test DNA may be any small DNA cut to completion with the same
restriction enzyme used to prepare the vector. Incubate 2 hours at room
temperature.
10. Dilute ligations and transform as in step 5 above.
11. Spread various amounts (e.g. 4 ul, 40 ul and 400 ul) of the transformation
mixture on LB agar plates containing 12.5µg/ml of chloramphenicol, 50µg/ml
Xgal and 25 µg/ml IPTG, and score the colonies after 18 hours of growth and
color development.
12. If the final concentration of the vector is too dilute, an ethanol
precipitation and resuspension in smaller volume can be performed at this
point.
13. Store the vector at -20' C. Aliquots at the appropriate dilution may be
stored at 4 ûC for over a month.
PREPARATION OF SOURCE DNA
A. Sources of DNA for BAC cloning
High molecular weight DNA used for BAC cloning is usually prepared in agarose.
B. Partial Digestion of Source DNA
C. Preparative PFGE
1. To cast the gel around the sample plugs, melt a solution of 1% Low Melting
Temperature Agarose gel in 1X TAE buffer and cool it to 45' C. The agarose
should be pure enough to permit subsequent enzymatic steps, e.g. SeaPlaque
GTG, (FMC BioProducts). TAE is used for preparative gels due to the
increased activity of the enzyme agarase in Tris-acetate, as opposed to
Tris-borate buffers.
2. Place the sample plugs on a comb, immediately next to each other (the
samples should be touching each other and without gaps, spanning any
gaps between teeth in the comb). Flank the samples with size markers,
such as yeast chromosomes (S. cerevisiae) and bacteriophage lambda DNA
ladders.
3. Position the comb on the casting tray, gently push the slices down to
touch the bottom of the casting tray, and seal the samples to the comb
by slowly pipetting a little bit of the cooled agarose solution over them.
4. When the plugs are set in place, pour the remaining agarose in the tray,
using just enough to completely submerge the plugs. Avoid casting a very
thick gel that would lead to dilution of the sample after melting.
5. Let the agarose harden for approximately 1 hour in the cold room, carefully
slide the comb out and gently position the gel in the newly cleaned gel box.
Electrophoretic conditions will vary with the size range desired for the
separation. Conditions that provide separation of fragments from 50 kb to
over 800 kb are: 1x TAE, 1% low melting agarose, fields of 6 V/cm with
a 120û reorientation angle and 90 second switching at 14' C for 18-22 hours.
6. After electrophoresis, remove the gel to a solid support and using a new
scalpel or razor blade, slice off the outer portions of the gel, including
the marker lanes and approximately 4 mm from each side of the sample.
7. Stain these outer portions of the gel with ethidium bromide while leaving
the rest of the gel covered with plastic wrap.
8. Photograph the stained portions including clean rulers on each outer edge
to indicate distance. This will permit the desired size range to be located
on the unstained central portion of the gel. The size distribution of the
partially digested DNA fragments observed in the stained outer portion of
the gel should be consistent with that predicted by the titration of partial
digestion conditions from the pilot experiments.
9. Place clean rulers next to both edges of the unstained central portion of
the gel to correspond to the positions depicted in the photograph. Lay a
third clean ruler or straight edge horizontally across the top of the gel
to act as a guide when cutting each region across the entire width of the
gel, containing the different sized DNA fractions.
10. Slice across the gel every 5 mm, producing long agarose "noodles" containing
size fractionated DNA, with dimensions of approximately 0.5 x 0.5 cm x the
length corresponding to the width of the sample that was loaded. Typically,
for gels run using 1x TAE, 1% low melting agarose, fields of 6 V/cm with a
120û reorientation angle, and 90 second switching at 14 ûCfor 18-22 hours,
each 5 mm noodle will contain DNA spanning a range of about 75 kb.
11. Using the photograph as a reference, save fractions starting from those
at an apparent size of 100 kb up to those over 800 kb. Mark on the
photograph where slices were made.
12. Cut a slice of about 2mm off the end of each fraction for analytical PFGE.
Keep these slices in their proper order by placing the 2 mm sample
immediately on the comb or in the well of the analytical gel.
Separate these samples using electrophoresis conditions similar to those
of the original preparative gel. The sizes of the DNA will often appear
smaller than that predicted based on the first preparative gel.
In addition, the DNA which had previously migrated in a 0.5 cm region of
the gel will now show a smear over a much larger region (1 to 3 cm).
13. Store the sized DNA fractions in 15 ml tubes containing 50mM EDTA.
Label these tubes using the same nomenclature as that used to label the
photo. Store the fractions at 4' C, where they are stable for several months.
Second preparative sizing gel. When the size of BAC clones is smaller than
expected the reason may be that smaller fragments comigrated witht he desires
fragments during the preparative electrophoresis. In such cases, which often
reflect excessive DNA concentrations, a second size selection may be effective.
1. Based on the results of the analytical gel of the small portions of the
samples run analytically, select a fraction of interest for cloning. The
criteria include both size (e.g. see table ?) and sufficiently high DNA
concentration to withstand subsequent dilution upon running in a second gel.
2. Place the entire slice, or noodle, flat against the comb, flanked by size
markers. Seal these to the comb with agarose as described before.
3. Cast and run the gel as described above.
4. Slice off the size markers and an aaproximately 4 mm portion from each side
of the sample, stain and photograph . During this second sizing step, the DNA
will usually have spread over an area of about 1- 2 cm.
5. Once again, excise the DNA from the preparative portion of the gel, slicing
every 5mm in the region of interest, as guided by the photo and clean rulers.
6. Label the fractions to correspond to the photograph and store at 4û C in 0.05M EDTA.
These are the fractions that will be used for cloning.
D. Recovery of DNA from gels and protecting the large DNA.
Size selected DNA can be recovered from gels using the enzyme agarase. Agarose is
melt before the addition of agarase to facilitate the digestion of agarose fiber by
the enzyme. Large linear DNA molecules are highly vulnerable to mechanical shearing
once they are released form agarose matrix, and therefore it is beneficial to include
positively charged polymers such as polyamines to compact and stabilize the DNA.
Maintaining the DNA in 50 mM NaCl throughout the procedure is recommended also.
E. Determining the concentration of gel purified DNA
1. Cast a mini-gel (approx. 5 x 8 cm) of about 2 mm thickness using 1 % agarose
in 1 X TBE. The teeth of the comb used should be less than 4 mm wide.
2. Mix 10ml of the agarase digested sample (from step # 6 in Ligation protocol
below) with 2 ml loading dye. A regular (narrow opening) pipet tip can be used,
since DNA breakage will not interfere with the results.
3. Run the sample, using a dilution series of intact bacteriophage lambda DNA
(for example 1.66ng, 5 ng, 15 ng and 45 ng) or bacteriophage lambda DNA
digested with HindIII (for example, 5 ng, 15 ng, 45 ng, and 90 ng) as
standards. Run the gel using a high voltage gradient (approx. 7-10 V/cm)
for 15 to 30 minutes. Stain the gel in ethidium bromide for 5 minutes to
visualize the bands. Photograph and estimate the insert DNA concentration.
LIGATION AND ELECTROPORATION
A. BAC LIGATION
1. Slice 3-5 mm from the end of the size fractionated sample of DNA in low
melting agarose.
2. Equilibrate the sample by transferring it to a new polypropylene tube
containing 50 ml TE+50mM NaCl and gently rocking at 4û C for 2 hours.
Carefully pour off the buffer, replace it with fresh solution, and equilibrate
for an additional 1-2 hours. Decanting is greatly facilitated by use of
screen caps (BioRad Labs cat. # 170-3711) on 50 ml conical bottom centrifuge
tubes.
3. Transfer the solid sample to a 1.5 ml microfuge tube, blotting away excess
buffer with the tip of a tissue.
4. Heat the sample at 65û C for 10-15 minutes to melt the agarose. After
inspecting to make sure that the agarose is thoroughly melted, transfer the
tube to a 45û C water bath and equilibrate the sample by incubating it there
for three minutes.
5. Add Gelase (or beta-agarase ), using 1.5 units /100µl molten agarose. Mix
by stirring gently with the pipet tip and incubate at 45û C for 1 hour.
6. Spin the sample in a microcentrifuge for 3 seconds to sediment undigested
agarose, transfer the supernatant to a new tube using a wide bore tip.
Discard the tube with any sedimented agarose, and place the tube on ice.
7. Ligate the sample with the molar ratio of vector to insert established
in the test ligations (described above) which is likely to be between 5:1
and 10:1. This requires an estimation of both the concentration of the
DNA and its average length. The volume of the ligation will be dictated
by the insert DNA concentration. Add the other components in a minimal
volume. For example, assuming that the insert DNA in agarose is approximately
1 ng/ul, and is on average 300 kb, a typical ligation would be: 40 ul insert
DNA (40 ng of average length 300 kb), 6 ul 10X ligation buffer (supplied by
the enzyme supplier), 0.6 ul pBeloBAC vector (containing 9.2 ng) (7.4 kb
length), 4 ul DNA ligase diluted in 1X ligation buffer (containing 40 NEB
units), 9.4 ul water, 60 ul total.
Note:
To free any sticky ends that may have annealed during preparation or storage
of the DNA, the insert and vector DNA should be heated briefly and cooled just
prior to the addition of the buffer and enzyme. Using a wide bore pitpet tip,
transfer the DNA to a microfuge tube, add the vector and water. Heat to 65û C
for 4 minutes. Cool briefly at room temperature, before adding 10 X ligation
buffer (containing ATP), and ligase. Mix by gently stirring with the pipet tip.
8. Ligate overnight at 16û C.
9. Centrifuge in a microfuge for a few seconds to collect the sample. BAC
ligations should be transformed soon after this. A significant decrease in
transformation efficiency is detectable within 2 days after ligation.
10. Dialyze the sample prior to electroporation using a drop dialysis
technique (also referred to as spot dialysis), as described below.
11. Transfer the sample to a microfuge tube and place on ice.
Drop Dialysis: Drop dialysis is a fast way to remove salts or other small
molecules from small volume samples. The sample, from 20 to 400 ul, is placed on
top of a filter that is floated on buffer in a Petri dish.
1. Add 20-30 ml of 0.5X TE to a 100 mm Petri dish. Place the Petri dish where
it will be undisturbed by contact or vibration.
2. Using blunt forceps, gently place a filter (Millipore cat # VSWP 02500) on
top of the solution with the shiny side facing up. If more than one sample
will be dialyzed simultaneously, the filter should be marked with pencil or
water proof pen prior to application of the sample. Allow the filter
approximately one minute to fully wet.
3. Slowly pipet the sample onto the surface of the filter, using a wide-bore
pipet tip. Cover the dish and allow to dialyze from 20 minutes to two hours
at room temperature.
4. Remove samples with a wide-bore pipet tip, expect to recover about 90% of
the sample volume. Do not try to remove the entire sample, since this is
likely to submerge the filter with resulting loss of all remaining samples.
Notes:
Up to four samples of 50 ml each can be placed on a single filter. Brief
dialysis, less than 1 hour, may increase the sample volume if the applied
sample contains a high concentration of salt or sucrose. Prolonged dialysis
(more than 4 hours) will result in loss of sample volume due to gravity.
B. Electroporation of Large DNA
1. Preparation of electrocompetent cells
Preparation of Electrocompetent Cells
[This procedure is based on that provided by Joel Jessee of Life Technologies,
Gaithersberg MD. It will provide approximately 2 ml of competent cells,
sufficient for approximately 60-80 transformations.]
1. Inoculate a 3 liter flask containing 0.8-1 liter of SOB
(without Magnesium) using a fresh overnight saturated culture
of DH10B at a dilution of 1:1000.
2. Grow the cells at 37û C shaking them at approximately 200 rpm.
Monitor the cell growth by reading the optical density at 550 nm.
3. Harvest the cells when the OD reaches between 0.6 and 0.8. This
usually takes approximately 5 hours, though the use of pre-warmed
medium can shorten this by about 1 hour. It is important that the
cells be harvested while still in log phase, i.e. they should not
be allowed to reach an OD600 > 0.8.
4. Transfer the culture into centrifuge bottles and centrifuge at 5000 rpm
(4,000 x g) for 10' at 4û C. The rotor should be prechilled.
5. Resuspend the cell pellets in a volume of ice cold 10% glycerol equal
to the original volume of the culture.
6. Centrifuge at 5,000 rpm for 10' at 4û C.
7. Carefully pour off the supernatant (this time the pellet will be quite
loose) and again resuspend the cells in an equal volume of ice cold
10% glycerol to wash the cells a second time. Collect the cells by
centrifugation at 5,000 rpm for 10' at 4û C.
8. Pour off the supernatant, and resuspend the cells in the small volume
that remains in the bottles. Transfer the cell suspension to a single
centrifuge tube (50 ml Nalgene).
9. Centrifuge again at 4û C for 10' at 7,000 rpm. This last centrifugation
is done to facilitate the resuspension of the cells in a small volume
by collecting them in a smaller and tighter pellet.
10. Pour off the supernatant and resuspend the cells in approximately 2 ml
of ice cold 10% glycerol per liter of initial culture.
11. Distribute the cells in convenient aliquots into microfuge tubes
(25-30 ml will be used per transformation) and quick freeze them in a
dry ice-ethanol bath.
12. After 5' transfer them to -70û C for storage until the moment of use.
13. Thaw an aliquot of cells and streak them on LB agar to produce single
colonies to examine the preparation for possible contamination (which
would appear as unusual colony morphology phenotypes).
Notes
1. During the preparation, keep the cells on ice when they are not in the
centrifuge. They should be promptly removed from the centrifuge at the
end of each spin.
2. SOB is a very rich medium, and promotes rapid growth of any contaminating
microorganism. Since even trace amounts of this contamination which may
escape visual detection will spoil a batch of cells, test the sterility
of the SOB by incubating it at 37û C overnight prior to use.
3. The transformation efficiency of these cells with small DNA (plasmids of
a few kb) should be greater than 109 colonies per microgram.
4. Thawed cells can be refrozen for use at a later date. There is not a
significant drop after a second round of freezing.
2. Electroporation of BAC ligation mix
Prior to electroporation, ligations must either be dialyzed or diluted to prevent the
sample from causing an arc to jump across the cuvette upon application of the pulse.
We prefer dialysis as described in section IV A. above, since dilution increases the
number of electroporations that must be carried out.
1. Aliquot 0.5ml SOC into sterile culture tubes, one tube for each DNA sample.
2. Place the electroporation cuvettes, either 0.1 or 0.2 cm gap, on ice.
3. Allow the competent cells to thaw on ice. Then aliquot 25µl cells for each
DNA sample into a microfuge tubes. Leave the tubes on ice.
4. Set the desired conditions on the electroporation apparatus. To generate
BACs greater than 80 kb, use the following optimized conditions (Sheng et al.
1995): Capacitance 25 microfarad, resistance 100 Ohms and a voltage gradient
of either 12.5 or 9 kV/cm.
5. Add 3 µl of the dialyzed ligation mixture to the tube containing the cells,
flick the tube with a finger to mix, and transfer the cells to the bottom of
the electroporation cuvette. Without delay, wipe the outside of the cuvette
with a tissue to dry it, place it in the electroporation chamber and apply
the pulse.
6. Immediately after pulsing, add the pre-measured 0.5 ml of SOC to the cuvette
with a Pasteur pipette or disposable narrow plastic transfer pipet and
transfer the cell suspension back into the culture tube. Delaying this
transfer can seriously reduce the survival of transformed cells.
7. Transfer the tube to 37û C and grow for 45 minutes while shaking at 200 rpm.
This length of time has been chosen to allow full recovery and expression of
the Chloramphenicol resistance gene while avoiding doubling of the cells,
which would give rise to unwanted duplicate clones.
8. Spread the cells on LB Agar plates containing 12.5µg/ml of chloramphenicol,
50µg/ml Xgal and 25 µg/ml IPTG. Plating is described in detail in section C
below. The samples can be spread using a glass rod or autoclaved glass beads.
9. Incubate the plates at 37û C for at least 18 hours, to permit the color to
develop sufficiently to distinguish blue colonies from white. If the colonies
will not be picked immediately, store the plates at 4û C.
Notes
1. When transforming a large volume ligation, it is best to pool all the cells
after the 45 minute recovery period and then plate from this pooled material.
Alternatively, transformed bacteria may be combined prior to the 37û C incubation.
For large scale transformations (those involving ligations of more than 75 ml)
it is extremely useful to have two people participate in the electroporation
and spreading process, to avoid delays that would diminish bacterial viability.
C. Plating and picking of BAC transformants
BAC clones are grown on LB agar plates containing 12.5-20 µg/ml chloramphenicol to
select for maintanence of the BAC. X-gal and IPTG are included for color selection.
Colony color will continue to develop on storage of the plates at room temperature
or at 4' C, but usually 24 hours is sufficient.
MANIPULATION AND ANALYSIS OF BACS
A. Purification of BAC DNA via mini-preps
1. Inoculate a colony into a 10 ml culture tube containing 2 ml
LB+ 12.5µg/ml chloramphenicol.
2. Grow overnight at 37û C by shaking at 200 rpm.
3. Transfer 1.5 ml of the culture to a 1.5ml microfuge tube.
4. Centrifuge at full speed in a microfuge for 30 seconds to collect the
cells. Aspirate or pour off growth medium.
5. Thoroughly resuspend the cell pellet in 100µl chilled Solution I by
pipetting.
6. Place the tubes on ice and add 200µl of freshly prepared Solution II.
Cap the tube, mix by inversion 8-10 times and return tubes to ice.
At this stage the cells will lyse and the bacterial suspension will
become clear and viscous.
7. Add 150µl of Solution III. Cap tube, mix by inversion 8-10 times and
return to ice. The addition of Solution III will cause the formation of
a flocculent precipitate.
8. Centrifuge at room temperature for 6 minutes at full speed in a microfuge.
9. Transfer the supernatant by pipetting or pouring to a new microfuge tube.
Any visible debris that is transferred can be removed with a toothpick
or pipet tip.
10. Precipitate the DNA by adding 1 ml room temperature 100% ethanol and
mixing by inversion.
11. Centrifuge at room temperature for 6 minutes in a microfuge.
12. Pour off the supernatant and rinse the pellet by adding 500µl of room
temperature 70% ethanol. Avoid dislodging pellet.
13. Pour off the ethanol and drain the tube by resting it upside-down on a
paper towel. Allow to dry completely (incubation at 37û will hasten the
drying).
14. Redissolve in 20µl TE. Allow DNA to rehydrate for 1-3 minutes in TE
at room temperature. Pipet the solution up and down a few times to get
the DNA into solution.
NOTE: Contaminating E. coli DNA can be efficiently removed by using ATP-dependent
DNAse (commercially available from Epicentre as "Plasmid-Safe DNAse") that
selectively digests linear DNA leaving supercoiled DNA intact.
B. PFG separation and purification of BACs
1. Direct electrophoresis of uncut BAC DNA
1. Prepare DNA by the mini-prep method described above starting with
1.5 ml culture volume.
2. Resuspend the DNA in 10 ml TE.
3. Mix 5 ml DNA with 1 ml loading dye.
4. Apply to an 0.8% gel in 0.5X TBE.
5. Run for 20 hours at 14û C using a voltage gradient of 6 V/cm and
reorientation angles of 120û, with a switch time of 45 seconds.
Notes
1) The switch interval is not critical to the effectiveness of the separation,
and a range of anywhere from 10 to 90 seconds will give nearly identical
separation of these supercoiled DNAs.
2) Size standards for large supercoiled DNAs consist of BAC DNA samples from
clones of known sizes.
3) For size analysis of supercoiled DNA, the samples must be analyzed within a
few days after preparation. Mini-prepped BAC DNA samples contain sufficient
amounts of nuclease to cause nicking of the DNA on storage even at 4û C. Also,
rough physical treatment of the samples, for example vortexing, will cause
large supercoiled BACs to become nicked. Nicked large circular DNA will remain
trapped in the well, leading to a lack of detectable bands on electrophoresis.
4) The use of separation times greater than 24 hr, has been found to produce broad
bands that often make detection with ethidium bromide impossible. Field
inversion gel electrophoresis has not been found effective for resolving these
molecules.
2. Excision of BAC inserts with NotI
1. Purify BAC DNA using the alkaline lysis mini-prep technique above. DNA should
be used for NotI digestion within 2 days of preparation to minimize degradation
of the DNA. Resuspend the DNA obtained from 1 to 3 ml of a saturated culture
in 20 µl TE by pipetting up and down.
2. Digest 5µl of the resuspended DNA in a reaction of at least 20µl using 3 units
of NotI. This is accomplished by preparing a fresh mixture containing the buffer,
enzyme, and water, and adding 15 µl of this mix to 5µl DNA. For example, mix the
following: 2 µl 10X NotI buffer, 12.7 µl water, 0.3 µl NotI (10U/µl),
add 5µl dye.
3. Incubate at 37û C for 1 hour.
4. Add 5 µl loading dye, and apply the entire reaction to a single lane of a 1%
agarose gel.
5. A 17 hour run using the following conditions gives excellent separation for
DNA molecules up to 250 kb, while keeping the BAC vector band still on the gel.
0.5 X TBE, at 14û C with a voltage gradient of 6 V/cm, a reorientation angle of
120û, and the switch time ramped linearly from 5 to 12 seconds.
3. Interpretation of NotI digests.
The intensity of ethidium bromide fluorescence in the vector band should be relatively
uniform from lane to lane. If a lane shows no visible vector band, and a large band of
equal intensity to those large bands in other lanes, it should be assumed that the
digestion was incomplete. Also, since ethidium fluorescence is directly proportional to
the mass of the DNA in each band, if multiple bands are present in a lane, yet the
difference in their intensity does not correspond to their apparent size, partial
digestion should be suspected. BACs which appear to contain no visible insert bands
may be presumed to contain either insert fragments which are smaller than the vector
and will have run off the gel or are just not visualized with ethidium, or vector
without inserts, in which the cloning site was altered to give rise to a white colony
color (the frequency of these clones should be known from the control ligations).
4. Isolation of BAC DNA from pulsed field gels
Recovery of BAC DNA from pulsed field gels using agarase.
1. Digest mini-prepped DNA obtained from approximately 1.5 ml of saturated
cells in a reaction volume of 50 microliters. To obtain intact large
fragments such as with NotI digestion, the DNA must be no more than a
few days old to avoid degradation by nuclease present in the DNA sample.
For NotI digestion use 8-10 units of enzyme and digest at 37'C for 1.5 hour.
Digestions with EcoRI (and other enzymes which are less sensitive to
inhibition by components present in mini-prepped DNA) may be carried out
in smaller reaction volumes.
2. Separate digests on 1% preparative pulsed field gels using low melting
temperature agarose. Agarase is approximately 10X more active in the
presence of Tris-acetate buffer, than Tris-borate. Therefore, use of
TAE (instead of TBE) will allow use of less enzyme for digestion of the
agarose, or will eliminate the need to exchange the buffer in the gel slice
prior to agarase treatment.
3. For NotI digests, run the gels in TAE using a reorientation angle of 120û
at 14' C at 6 V/cm for 17 hours ramping the switch time from an initial
value of 5 seconds to a final value of 12 seconds.
4. Stain the gel with Ethidium bromide, and excise the bands of interest,
exposing the DNA only to the minimum dose of UV light necessary. Place
the DNA slices into pre-weighed 1.5 ml microfuge tubes. Weigh again,
to determine the volume of sample present. Alternatively, if the gel
was run in TBE buffer, place the DNA sample in a 15 ml tube and dialyze
by equilibrating in at least 5 ml 1 X TAE.
5. Heat the tubes to 65û C for 5 minutes to melt the agarose. Transfer the
samples to a 44û water bath, and allow to equilibrate at that temperature
for 1 minute.
6. Add 1 U Gelase (Epicentre) or b-agarase (New England Biolabs) per 100 ml
gel. Mix by gently stirring with the pipet tip, and incubate for 1 hr.
at 44' C. Check to see that there are no visible pieces of agarose
remaining in the tube. If large pieces of agarose are present, further
incubation at 44' C for 30 minutes after the addition of 0.5 U agarase
should be sufficient to remove them.
7. Add a volume of 1M ammonium acetate equal to that of the digested gel
and mix by inverting the tube.
8. Precipitate the DNA by addition of ethanol, using twice the combined
volume of gel plus acetate, followed by mixing. Spin at room temperature
for 30 min. at full speed in a microfuge.
9. Very carefully remove the supernatant from the pellet using a yellow
pipet tip. The ethanol must be drawn off slowly to avoid dislodging the
DNA.
10. Add 1 ml of 70% ethanol, and invert the tube once to rinse. Centrifuge
at room temperature for 10 minutes at full speed in the microfuge.
Remove the supernatant carefully (as above) and dry the DNA.
11. Rehydrate the DNA by adding 10 ml TE and allowing to sit at room
temperature for 1-3 minutes. Dissolve the DNA by pipetting. The DNA
will go into solution rapidly.
12. Check the DNA concentration by running 3 ml of this sample on a quantitative
conventional mini-gel (PFG not necessary-see section ? above).
REFERENCES
Gnirke, A., Huxley, C., Pterson, K., Olson, M.V. 1993 Genomics Microinjection of
intact 200- to 500-kb fragments of YAC DNA into mammalian cells. 15: 659-667.
Grant, S., Jessee, J., Bloom, F., and Hanahan, D. 1990. Differential plasmid rescue
from transgenic mouse DNAs into Escherichia coli methylation-restriction
mutants. Proc. Natl. Acad Sci 87: 4645-4649.
Kim, U.-J., Shizuya, H., de Jong, P., Birren, B. and Simon, M. 1992. Stable
propagation of cosmid sized human DNA inserts in an F-factor based vector.
Nucl. Acids Res. 20: 1083-1085.
Kim, U.-J., Shizuya, H., Birren, B., de Jong, P., Slepak, T., and Simon, M. 1994.
Selection of chromosome 22-specific clones from human genomic BAC library
using a chromosome specific cosmid library. Genomics. 22: 336-339.
O'Connor, M., Pfeifer, M. and Bender, W. 1989. Construction of large DNA segments
in Esherichia coli.Science 244: 1307-1312.
Sambrook, J., Fritsch, E. and Maniatis, T. 1989. Molecular Cloning: A Laboratory
Manual., 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor. New York.
Sheng, Y, Mancino, V. and Birren, B. 1995. Transformation of E. coli with large DNA
molecules by electroporation. Nucl. Acids Res. 23: 1990-1996.
Shizuya, H., Birren, B., Mancino, V., Slepak, T., Tachiiri, Y., Kim, U.-J., and
Simon, M. 1992. Cloning and stable maintencance of 300 kb fragments of
human DNA in Escherichia coli using and F-factor based vector. Proc. Nat.
Acad. Sci. 89: 8794-8797.
Wang, M, Chen, X-N., Shouse, S., Manson, J., Wu, Q-Z., Li, R., Wrestler, J., Noya, D.,
Sun, Z-G., Korenberg, J., Lai, E. 1994. Construction and characterization of
a human chromosome 2 specific BAC library. Genomics 24: 527-534.
Wang, M. and Lai, E. 1995. Pulsed field separations of large supercoiled and
open-circular DNAs and its application to bacterial artificial chromosome
cloning. Electrophoresis 16: 1-7.