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Fulton TM, Chunwongse J, and Tanksley SD. (1995) Microprep Protocol for Extraction of DNA from Tomato and other Herbaceous Plants. Plant Molecular Biology Reporter 13 (3): 207-209.

1. Collect 50-100 mg (approximately 4-8 new leaflets, up to 1.5 cm long) from a 1-3 week old tomato seedling(1) and nestle loosely in the bottom of a 1.5 ml Eppendorf tube (2).

2. Prepare fresh microprep buffer (see recipe below), keep at room temperature.

3. Add 200 ul of buffer and grind tissue with power drill and plastic bit (rinsing pestle with water between samples); add another 550 ul of buffer and either vortex lightly or shake entire rack by hand.

4. Incubate in 65C waterbath for 30-120 minutes.

5. Fill the tube with chloroform/isoamyl (24:1). Mix well. (This can be done by vortexing each tube or sandwiching tubes between two racks and vigorously inverting or shaking up and down 50-100 times).

6. Centrifuge tubes at 10,000 rpm for 5 minutes.

7. Pipet off aqueous phase into new microfuge tubes. Add 2/3-1 times the volume of cold isopropanol to each tube. Invert tubes until DNA precipitates.

8. Immediately spin at 10,000 rpm for 5 minutes (no more), pour off isopropanol and wash pellet with 70% ethanol (3).

9. Dry pellet by leaving tubes upside down on paper towels for approximately 1 hr or placing on sides in seed dryer for 15 minutes (longer if necessary).

10. Resuspend DNA in 50 ul of TE at 65C for 15 min.

11. Spin 10 min at 10,000 rpm, store at 4C for up to 1 week, -20C for longer storage.

12. For RFLP use, digest 15-25ul for one Southern blot (can expect 5-10ug DNA, use 15-20 units of enzyme). For PCR, use 1 ul.

1) If only PCR is needed, can use as little as 1 cotyledon, resuspend in 50 ul in Step 10 but use 5 ul to PCR
2) Tissue can be harvested and kept at room temperature for up to 3 hours or stored at 4C for up to 3 days
3) Can stop here, storing pellet in 70% EtOH at -20C indefinitely


General Notes:
Yield should be approximately 10-20 ug of DNA, enough for 2-4 Southerns or 50-100 PCR reactions. The number of samples can be maximized by using 2 drills with drill stands concurrently and a foot pedal on/off switch. One experienced person can do up to several hundred samples in one day. This protocol is known to work on tomato, pepper, apple, tobacco, strawberry, and artichoke.

Materials and Solutions required
Drill: Heavy duty household drill with keyless chuck (so pestles can be easily replaced)and plastic drill bit/pestles (VWR Scientific, catalog#KT95050-99)
Microcentrifuge: with a fixed angle rotor, capable of 10,000 rpm and holding as many samples as possible

DNA Extraction Buffer: 0.35 M Sorbitol, 0.1 M Tris-base, 5 mM EDTA; pH 7.5

Nuclei Lysis Buffer: 0.2 M Tris, 0.05 M EDTA, 2 M NaCl, 2% CTAB
Sarkosyl 5%

Microprep Buffer: 2.5 parts DNA Extraction Buffer, 2.5 parts Nuclei Lysis Buffer, 1.0 part 5% Sarkosyl. Add .3-.5g Sodium Bisulfite/100 mls buffer immediately before use (can be increased to avoid color in final product).

For 75 extractions:
25 mls DNA Extraction Buffer
25 mls Nuclei Lysis Buffer
10 mls Sarkosyl
60 mls Microprep Buffer, add .2g Sodium Bisulfite


     
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Troubleshooting guide
           
     
Problem Possible solution
Low concentration of DNA Grind longer
Use younger tissue
DNA does not form firm pellet After spin, remove replace 500ul isoprop. with 70% EtOH, gently mix, respin
DNA does not digest Dry pellet longer before resuspension (remove alcohol residue)
Take only the aqueous layer of the chloroform gradient, do not take any interface (avoid chloroform residue)
DNA does not PCR See solutions for not digesting
Use different amount of DNA (probably less)