Shorty DNA Prep (per Adam 08/12/04)【University of Chicago】

There are several ways to prepare DNA from plants. I have found this method to be extremely efficient and quick. The DNA prepared via this protocol can be used in any PCR reaction. However, the DNA from this prep can not be used for southern blots. Anywhere up to 180 samples can easily be prepared in one day. When doing this prep, it is useful to do an even number of samples (to balance the centrifuge) or a multiple of 30 (the number of positions in a large centrifuge) when handling a larger set.

1. Add a small amount of silica beads (about 300µl) to an eppendorf tube containing tissue sample. For a higher yield of DNA , use an entire seedling and for a mediocre amount use a leaf. This protocol is very sensitive and has been successful in isolating DNA from less than 10µg of tissue. Cover the tube with a cap with a hole and lyophilize (at least 4hours, overnight for larger tissues and better results).

2. Blast the dried leaf samples by vortexing for 10minutes or until the tissue has been completely macerated.

3. Add 500µl Shorty Buffer (recipe below) and 500µl PCI (Phenol:Cloroform:Isoamyl Alcohol at a ratio of 25:24:1), vortex. This step can be completed on a bench top with caution or in a fume hood.

NOTE: When handling phenol, it is necessary to wear appropriate protective gear (goggles, gloves, etc.).

4. Spin 10minutes at 8,000 rpm in micro-centrifuge.

5. Transfer 400µl of supernatant to a fresh eppendorf containing 400µl isopropanol.

6. Mix by inversion and spin 10minutes at top speed in micro-centrifuge.

7. Pour off liquid and add 300µl of 70%ethanol.

8. Mix by inversion and spin 5minutes at top speed in micro-centrifuge.

9. Pour off liquid and dry pellet by letting it sit upside down on a paper towel for 1hour.

10. Once the tube is dry, add 100µl TE (1X I would not use 10X because it can carry over salts that can potentially disrupt the reactions in which the DNA is going to be used) and resuspend DNA by vortexing at room temp for 5minutes, however this will shear the genomic DNA .

NOTE: If you do not want the DNA sheared let the tube sit at room temperature overnight.

Shorty Buffer (500mL) Glycogen (10mg/L) 1M Tris-HCl, pH 9.0 2M LiCundefined 0.5M EDTA 10% SDS H2 0

500µl 100ml 100ml 25ml 50ml 225ml

8.48 Lithium Chloride 100mL H₂ 0 = 100mL 2M LiCl