plasmid rescue protocol

[Randall L Scholl]

Protocol

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. 
(Fax  814-865-9131)

Key Words: Arabidopsis, plasmid rescue, T-DNA mutagenesis

Abstract: 

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.
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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
glucose
sterile deionized water
10% sterile glycerol
3 M sodium acetate, pH 5.2
100% ethanol
70% ethanol
phenol/chloroform: phenol equilibrated with TE: chloroform
     (1:1)
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
     by inversion.

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
     dissappeared.

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
     water bath.

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
     efficiency3.

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
h.

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
rapid shaking.

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
for reference.  

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
bromide ml-1.

Notes

1.  This is a summary of the method described by Eichholtz et
     al. (1987)

2.  Electroporation competent cells were prepared as
     described by Dower et al. (1987), with minor
     modifications.

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
     transformation.

5.  We generally recover 100-300 colonies per
     electroporation, though this number may sometimes be
     much lower.


     
               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
be 6