UBL Protocol

for

DNA Extraction ñ Plant Tissue - LN2 Sample Preparation

 

Revised May 2002

 

Part I:Ý Preparation of Mortars & Pestles (do this the day before you extract, preferably)

 

1.Ý Obtain enough mortars, pestles, and metal spatulas for your needs (1 set for each sample) and a pan to put them in.

 

2.Ý Soak the mortars (right side up), pestles, and spatulas in 10% bleach (e.g. dilute 100 mL Clorox with 900 mL water) for 15 minutes.

 

3.Ý Rinse everything (including the pan) thoroughly with DI water.

 

4.Ý Dry all components with paper towel, then line the bottom of the pan with fresh paper towel.

 

5.Ý Place the mortars (upside down) and pestles in the pan, then cover the pan loosely with aluminum foil.

 

6.Ý Place the metal spatulas in a paper or autoclave bag.

 

7.Ý Autoclave all components: sterilize for 30 minutes, slow exhaust for 10 minutes, then dry for 10 minutes.

 

8.Ý Allow the pan to cool, then place in ñ30C freezer overnight (do not freeze the spatulas).

 

 

Part II: Sample Extraction

 

1.Ý Be sure to have sufficient liquid nitrogen and the Qiagen extraction kit on hand before you start.Ý (Wear gloves and safety glasses when using liquid nitrogen!)

 

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

 

3.Ý Remove a cold mortar and pestle from the freezer (If extracting multiple samples, don't take them all out at the same time or they will warm up!)

 

4.Ý Carefully pour in liquid nitrogen to about ‡ full.

 

5.Ý Immediately place the sample in the mortar, then grind quickly and vigorously into a fine powder.Ý (Work fast-- if the sample sits for too long it will start to hydrate.Ý If that happens, carefully add more liquid nitrogen and repeat.)

 

6.Ý Using a sterile metal spatula, transfer the powder into a microfuge tube, then proceed immediately with Steps 2 & 3 of the Qiagen extraction protocol (400uL of AP1 and 4uL of RNase A, then 10 minute incubation)

 

Prepare the remaining samples the same way, then continue all samples with the Qiagen protocol at Step 4.

 

 

Part III: Cleanup (do this as soon as you are done with the extraction!)

 

Wash the mortars, pestles, and spatulas with soap and hot water, then rinse with DI water and let them air dry.Ý (If they sit for too long, they will be very difficult to clean!)