A rapid method of preparing megabase plant DNA. Fran9ois Guidet, Peter Rogowsky and Peter Langridge. Centre for Cereal Biotechnology, Waite Agricultural ...
Nucleic Acids Research, Vol. 18, No. 16 4955
k./ 1990 Oxford University Press
A rapid method
of
preparing
megabase
plant DNA
Fran9ois Guidet, Peter Rogowsky and Peter Langridge Centre for Cereal Biotechnology, Waite Agricultural Research Institute, The University of Adelaide, Glen Osmond, South Australia 5064, Australia Submitted January 29, 1990 Tris-HCl pH 8.0, 50 mM EDTA at 4°C or each plug is sliced in 5 pieces and incubated twice in 5-10 ml of the appropriate restriction buffer and very gently shaken at 30°C for 3-4 hours (this allows efficient change of buffer inside the plugs). The DNA is digested overnight in 100 u1 of restriction buffer at 37°C using about 50 units of restriction enzyme. The method has been successfully applied on wheat, tobacco and rye (Fig. lA). The isolated DNAs are larger than the largest DNA size marker (the chromosome XII of Saccharomyces cerevisiae is 2.5 Mbp long) and are fully digestible with restriction endonucleases (Fig. 1). The quantity of plant material can be easily scaled up to over 1g. This very rapid method has a high DNA yield and should be of particular interest to people who have experienced a low protoplast recovery with their plant material.
Recently published methods for the preparation of very large fragments of plant DNA suitable for pulsed field electrophoresis (1, 2) require the preparation of protoplasts from leaf material. In this report we describe a method which circumvents this tedious step and which, by its simplicity and efficiency, should appeal to a large number of researchers involved in long range mapping of plant chromosomes. The rationale of the method is as follows: when plant material is ground to a fine powder in liquid nitrogen, the cell wall is disrupted but the integrity of the cell organelles, including the nucleus, is maintained. This means that the DNA molecules remain intact. The powder is combined with low melting point (LMP) agarose and the resulting plugs digested in situ. Method: About 0.2 g of young leaves are ground to a fine powder in liquid nitrogen using a mortar and pestle. The powder is transferred to a crucible preheated to 50°C, mixed with 1 ml of 0.7% LMP agarose (in 10 mM Tris-HCl pH 7.8, 15 mM NaCl, 200 mM EDTA) and gently stirred with a sterile spatula to obtain a homogenous mixture (alternatively the powder can be mixed with 500 ,u of the agarose buffer and then added to 500 d41 of a 1.4% agarose solution). The mixture is poured directly into an opened mould (Biorad's CHEF-DR II mould) immediately cast and allowed to set at 4°C for 15 min. The plugs are then incubated twice at 50°C in 5 ml of 10 mM Tris-HCl pH 8.0, 500 mM EDTA, 1% Sarkosyl, 1 mg/ml proteinase K for 16-18 hours. Following this treatment they are either stored in 1 mM 1
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ACKNOWLEDGEMENT This work is supported by the Australian Research Council.
REFERENCES 1. Ganal,M.W., Young,N.D. and Tanksley,S.D. (1989) MoL Gen. Genet. 215, 395-400. 2. van Daelen,R.A.J., Jonkers,J.J. and Zabel,P. (1989) Plant Mol. Biol. 12, 341-352.
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1
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SIZE
(Kbp)
; E~~~~~~~~~~~~~~~
_~ ~~~~~~~
20
945 1125
-1020
B Figure 1. Pulsed field gel electrophoresis with a CHEF-DR II apparatus (Biorad); the electrophoresis is in 1% agarose gel. Panel A: 0.5 xTBE at 200 volts and 14°C with 60 second pulse time for 15 hours followed by a 90 second pulse time for 9 hours. Lanes are as follows: 1, 5, 7, undigested DNA from wheat, tobacco and rye respectively; 2, 6, 8, the same DNAs digested with HindHI (both digested and undigested DNAs are incubated overnight at 37°C); 3 and 4, yeast chromosome standards. Panel B: 1 xTAE at 150 volts and 14°C with a 100 second to 300 second ramped pulse time for 69 hours. Lanes are as follows: 1, non digested wheat DNA; 2, 3, 4, wheat DNA digested by SailI, Clal and SmaI respectively. All the plugs were incubated at 37°C overnight (except for the SmaI digestion: 25°C). Lane 5: yeast chromosome standards.
