UBL Checklist Protocol

for

DNA Extraction ñ Plant Tissue ‚ñ FastPrep

Please note: The checklist assumes that you are familiar

with basic techniques and just need to know what the steps are!Ý

Refer to the complete protocols if you need more information.

Revised May 2002

___ 1.Ý Weigh out 100 mg (0.1 g) of young leaf tissue.

___ 2.Ý Transfer the tissue into a 2 mL tube with º" ceramic bead and garnet matrix (provided with kit).

___ 3.Ý Add 800 μL of CLS-VF.

___ 4.Ý Add 200 μL of PPS.

___ 5.Ý Homogenize in the FastPrep Instrument for 20 seconds at speed 4^*.

ÝÝ ^* This is the standard setting for UBL isolations. Actual range is 5-30 seconds at a speed of 4-5.Ý Optimize as necessary

___ 6.Ý (optional) For dry tissues, incubate mixture for 15 minutes ñ 2 hours for improved DNA recovery.

___ 7.Ý Centrifuge at 14,000 x g for 5 minutes.Ý Extend spin to 15 minutes if debris does not pellet well. See figure below.

___ 8.Ý Transfer 600 μL of the supernatant (be careful here: debris at top and bottom!) into a clean microfuge tube.

___ 9.Ý Add 600 μL of Binding Matrix and mix gently.

___ 10.Ý Incubate at room temperature for 5 minutes.

___ 11.Ý Centrifuge for 1 minute, then discard the supernatant.

___ 12.Ý Resuspend pellet with 500 μL of SEWS-M.

___ 13.Ý Centrifuge for 1 minute, then discard the supernatant.

___ 14.Ý Centrifuge (pulse) for 10 seconds, then remove residual liquid with a small-bore pipet tip.

___ 15.Ý Resuspend pellet with 100 μL DES.

___ 16.Ý Incubate at room temperature for 2-3 minutes.

___ 17.Ý Centrifuge at 14,000 x g for 1 minute.Ý The solubilized DNA is now in the supernatant.

___ 18.Ý Transfer supernatant (avoid the pellet) to a new microfuge tube.

___ 19.Ý Label the microfuge tube and store samples in freezer.