Streamlined?DNA?Extraction?Protocol

This method is derived from a procedure developed by Toby Bradshaw and the Poplar Molecular Genetics Cooperative. We have tested the procedure with a variety of Populus species, as well as tobacco and Arabidopsis . The resulting DNA is of sufficiently high quality for PCR (including RAPD), restriction digests, and ligation reactions. However, it is extemely important to use the youngest leaves available for Populus , as DNA from older leaves is often contaminated with compounds that can interfere with enzymatic reactions. Expected yield is 5-30 μg, depending on the quality and quantity of the foliage. Yields can be increased beyond 30 μg by using multiple young leaves and increasing the volumes of the grinding and extraction buffers.

1.Add Diethyl Dithiocarbamic Acid Sodium salt (4 mg/ml) and RNAase A (100 μg/ml) to an aliquot of Grinding Buffer, and B-mercaptoethanol (1%) to an aliquot of Lysis Buffer (see below).

2.Use a single newly emerged, still rolled-up leaf (~10 mg, the newer the better). Place foliage in microfuge tubes in a liquid nitrogen bath.

3.Add approximately 200 to 400 μl of liquid nitrogen to a tube containing foliage. Grind approx. 10-15 seconds using a motorized pestle. Stop grinding when liquid nitrogen evaporates and tissue defrosts.

4.Add 200 μl Grinding Buffer. Grind another 10 seconds until tissue is well-homogenized.

5.Place tube in hot water bath (~40-65 deg.) and incubate while grinding other samples. Each sample should be incubated at least 10 min.

6.Add 200 μl Lysis Buffer to each tube. Mix by inverting several times.

7.After grinding all samples (typically as many as will fit in microfuge), incubate at 65 deg. for 30-60 min. Every 10 to 15 minutes, mix by inverting tubes several times.

8.Add equal volume of phenol:chloroform:IAA (25:24:1). Mix well by inverting at least 10 times. Spin 8-10 min. at 12,000 rpm at room temperature.

9.Remove 50-70% of supernatant to new tube, taking care to avoid interface. If supernatant is cloudy or contaminated with interface, go to step 10. Otherwise skip to 12.

10.(Optional) Add an equal volume of chloroform, mix well by inverting at least 10 times. Spin 5 min. at 12,000 rpm at room temperature.

11.Remove 50-70% of supernatant to new tube, taking care to avoid interface.

12.Add 2/3 volume of isopropanol. Mix by inverting.

13.Incubate 15-30 min. on ice or room temperature.

14.Spin in microfuge 5-10 minutes at room temperature.

15.Decant supernatant and blot tubes on kimwipe.

16.Dry in speed-vac for 2-4 minutes until no isopropanol odor is apparent.

17.Add 50 μl TE buffer (10:1), pH 8. Let sit at room temperature for 10-20 min., or at 4 deg. overnight (preferred). Mix by flicking tube several times.

Grinding Buffer

100 mM Tris pH 8

20 mM EDTA pH 8

4 mg/ml diethyl dithiocarbamic acid, sodium salt, added just prior to use.

100 μg/ml RNAase A, added just prior to use

Lysis Buffer

100 mM Tris pH 8

20 mM EDTA pH 8

1 M NaCl

2% SDS

1% B-mecaptoethanol, added just prior to use