plasmid rescue protocol
Randall L Scholl
rscholl at magnus.acs.ohio-state.edu
Mon Mar 30 16:07:58 EST 1992
A Plasmid Rescue Technique for the Recovery of Plant DNA
Disrupted by T-DNA Insertion
Friedrich J. Behringer and June I. Medford
Department of Biology, 512 Wartik Laboratory, The
Pennsylvania State University, University Park, PA 16802 USA.
Key Words: Arabidopsis, plasmid rescue, T-DNA mutagenesis
Arabidopsis mutants generated by insertion of the T-DNA from
Ti plasmid 3850:1003 serve as a starting point for the
isolation of novel genes. The disrupted plant DNA can be
recovered using a plasmid rescue technique utilizing high
efficiency electroporation. Rescued plasmids are resistant
to ampicillin and contain an origin of replication from
pBR322. Plasmids generated from either the left or right
border of the T-DNA that carry flanking DNA sequences can be
identified by analyzing the products of restriction enzyme
digests on agarose gels. The plasmids with flanking
sequences can then serve as a starting point for cloning
plant sequences that share homology to the DNA at the point
of T-DNA insertion.
Insertional mutagenesis is a general method of generating new
mutants and allows the cloning of novel genes. Mutagenesis
with the T-DNA of the cointegrate Ti plasmid 3850:1003 is
among one of the recent gene tagging strategies (Feldmann and
Marks, 1987) and has generated a number of novel mutants
(Feldmann, 1991), some of which have lead to the cloning of
genes that regulate development (Yanofsky et al., 1990;
Herman and Marks, 1991). Once genetic linkage between the T-
DNA and mutation is indicated, the next step involves cloning
the plant DNA that has been insertionally disrupted. In
principle a number of techniques, including library
construction and screening, inverse PCR, and plasmid rescue
can be used to obtain tagged plant DNA. Here we describe a
plasmid rescue approach to recovering T-DNA plasmids that
contain plant DNA.
Procedure Solutions and media:
plant DNA extraction buffer: 50 mM Tris, 50 mM EDTA, 50 mM
NaCl, 400 micro-g ml-1 ethidium bromide, 2% N-lauryl
sarcosine, pH 8.0
TE: 10 mM Tris, 1 mM EDTA (Na salt), pH 7.0
TBE (5 X): 0.45 M Tris, 0.45 M boric acid, 0.01 M EDTA
10 mM ATP
10 X ligation buffer:660 mM Tris,50 mM MgCl2,10 mM DTE,pH 7.5
LB medium: 1.0% bactotryptone, 0.5 % bacto yeast extract,
1.0% NaCl, pH 7.0
LB carb: 100 micro-g carbenicillin ml-1 LB
LB agar/carb: 15 g agar L-1 LB, add carbenicillin to a final
concentration of 100 micro-g ml-1 once the melted agar
solution has cooled to 50 C
S.O.C. medium: 2% bactotryptone, 0.5% yeast extract, 10 mM
NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM
sterile deionized water
10% sterile glycerol
3 M sodium acetate, pH 5.2
phenol/chloroform: phenol equilibrated with TE: chloroform
0.7% agarose: 0.7 % agarose (w:v) in 1 X TBE, contains 0.5
micro-l/ml ethidium bromide ethidium bromide stock
solution: 10 mg ml-1 ethidium bromide
l0 X loading dye: 0.25% bromophenol blue, 0.25% xylene cyanol
FF, 10 mM EDTA, 15 % Ficoll
Preparation of genomic DNA1
1. Thoroughly grind fresh plant tissue with a mortar and
pestle using 2-4
ml plant DNA extraction buffer per g tissue.
2. Centrifuge 30 min at 12,000 g, 5 C. Transfer the
supernatant to a fresh tube.
3. Add exactly 0.95 g CsCl per ml extraction buffer. Mix
4. Transfer the solution to Beckman polyallomer 13 X 51 mm
ultracentrifuge tubes. Centrifuge at 65 k RPM at 20 C
for 4 h with a VTi 65 rotor.
5. Remove the DNA band with a 16 gauge needle and transfer
to a microfuge tube. Add an equal volume of isoamyl
alcohol. Mix by inversion and centrifuge 1 min at RT at
maximum speed in a microfuge. Transfer the aqueous
phase to a fresh tube and repeat the extraction 3 times
or until all traces of ethidium bromide have
6. Dialyse the DNA solution against TE at 4 C.
7. Transfer the DNA solution to microfuge tubes. Add 1/10
vol 3.0 sodium acetate and 2 volumes 100% ethanol and
precipitate the DNA at -20 C.
8. Centrifuge 5 min at 14,000 RPM, 4 C. Wash pellet with
70% ethanol. Resuspend DNA in TE and quantitate by
measuring the A260/280.
Preparation of electroporation competent cells2
1.Inoculate four 1 L Erlenmeyer flasks containing 100 ml
LB growth medium with 1 ml of E. coli strain DH5-alpha
cells taken from an overnight culture. Grow cells to
an A600 of 0.300 - 0.400 with rapid shaking in a 37 C
2.Distribute the cell suspension to two 250 ml centrifuge
bottles and centrifuge at 2,500 g for 10 min at 4 C.
Decant the supernatant. Keep cells on ice hereafter.
3.Resuspend the cell pellets in 200 ml ice-cold sterile
water and centrifuge as before. Pour off the water.
4.Wash the cells in 200 ml ice-cold water a second time
and repeat centrifugation.
5.Resuspend the pellets in 50 ml of ice-cold 10% glycerol
and transfer to Oakridge tubes. Centrifuge at 2000 g
for 10 mi at 4 C.
6.Resuspend the cells in a total volume of 1.2 - 1.4 ml of
ice-cold 10% glycerol. Aliquot 200 micro-l volumes to
microfuge tubes, freeze on dry0ce and store at -80 C.
Cells stored for up to 1 month retained high
Rescue of T-DNA plasmids
1.In a microfuge tube, add 1-2 micro-g plant DNA, 2 micro-
l of the appropriate reaction buffer, water to 18 micro-
l and 10 U restriction enzyme (using Sal I for left
border rescues and EcoR I for right border rescues).
Incubate at 37 C for 2 h.
2.Bring volume to 200 micro-l with water. Add an equal
volume of phenol/chloroform and vortex. Centrifuge 1
min. Transfer aqueous phase to a fresh tube. Add 1/10
volume of 3.0 M sodium acetate and 2 volumes of 100%
ethanol and precipitate the DNA at -20 C for at least 1
3.Centrifuge 10 min at 4 C in a microfuge. Discard the
supernatant and wash the pellet with 70% ethanol.
Resuspend in 15 micro-l TE.
4.Add 2 micro-l 10 mM ATP, 2 mM 10 X ligation buffer and 1
micro-l (8 U) T4 ligase (Boehringer Manheim), to the Sal
I or EcoR I digested DNA. Incubate at 14 C for 12-24
h. Store the reaction mixture at -20 C.
5. Thaw the electroporation competent cells rapidly and
place on ice. Mix 1 to 2 micro-l of the ligation
reaction (100-200 ng) with 200 micro-l electroporation
competent E. coli and transfer the suspension to an ice-
cold electroporation cuvette (0.2 cm electrode gap, Bio
Rad Laboratories). Adjust the settings of the
electroporator ('Gene Pulser', Bio Rad Laboratories)
to provide an electric field of 12.5 kV/cm and an
exponential pulse decay time constant (tau or RC time
constant) of 8.2 ms (by adjusting the capacitance (C)
and resistance (R) to 21 micro-farads and 400 ohms ,
respectively). Electroporate the cells.4
6.Quickly transfer the cells to 10 ml warm S.O.C. medium
in a 100 ml Erlenmeyer flask and culture at 37 C with
7.After 1 h, spin down the cells in a 15 ml conical bottom
centrifuge tube with a clinical centrifuge.
8.Resuspend the cells in 0.3-0.4 ml S.O.C. and plate in 3
equal aliquots onto three 100 X 15 mm LB carb plates.
Incubate the plates at least 20 h at 37 C to allow
colonies growth. Store plates at 4 C.5
Analysis of carbenicillin resistant colonies
1. Prepare plasmid DNA from individual colonies using a
standard alkaline lysis minprep procedure. Streak
colonies selected for analysis on a fresh LB carb plate
2. For the analysis of left border rescues, digest the
miniprep DNA with Pst I. For analysis of right border
rescues digest with Eco R I and Sal I in combination.
Stop reactions with 1 tenth volume 10 X loading dye.
3. Electrophorese the digests on a 0.7% agarose gel in 1X
TBE running buffer containing 0.5 micro-l ethidium
1. This is a summary of the method described by Eichholtz et
2. Electroporation competent cells were prepared as
described by Dower et al. (1987), with minor
3. When properly prepared, these cells will give
transformation efficiencies > 5 X 108. High efficiency
is necessary for successful plasmid rescue.
4. We observed the actual tau to vary between 8 -10 ms.
Excess salt may result in arcing and negligible
5. We generally recover 100-300 colonies per
electroporation, though this number may sometimes be
Results and Discussion
The insertion of the T-DNA from Ti 3850:1003 in a plant's
genomic DNA provides the opportunity to isolate the T-DNA
borders along with flanking DNA. This is done by forming
chimeric plasmids between the inserted T-DNA and the adjacent
plant DNA. Sequences from the plasmid pBR322 provide an
origin of replication and the beta-lactamase gene allows for
selection with ampicillin. Two types of replicating
plasmids, internal and external, can be formed by the
ligation of Sal I or EcoR I digested genomic DNA from plants
with a T-DNA insertion (Fig. 1). Internal plasmids are
composed entirely of T-DNA. The Sal I internal plasmid will
More information about the Arab-gen