Extraction of Total DNA from Bermudagrass
Using
Qiagen’s QIAamp DNA Stool Mini Kit
Cat. No. 51504
Rational for using this kit for extraction of DNA: Oftentimes plant materials will contain
high amounts of inhibitors such as phenolics. These phenolics are frequently difficult to
remove from DNA extractions and can inhibit the PCR reaction. The above kit has been
optimized for removal of phenolics during the extraction process and has been found to
be useful (in spite of the “funny” name!)
A written protocol is included with each kit ordered and has pertinent information not
included here. Use this protocol for today’s Bermudagrass DNA isolation, optimized for
our centrifuges and situation.
Pre-heat water bath to 70ºC.
Sample selection:
1. Pre-weigh the screw cap 2mL bead-beater tube provided. Note: the bead beater
tube already contains two 6 mm glass beads. Be sure not to remove these.
Record the weight.
2. Select dark, discolored tissue from the Bermudagrass roots provided. You will
need about 100 mg of tissue (enough to fill the tube to the raised ring on the tube).
Break or cut into very small pieces (no more than 3/8” long) so that they fit easily
into the tube.
Tissue maceration:
3. Drop filled tube into liquid Nitrogen until bubbling stops.
4. Using long forceps, retrieve the tube in the beadbeater and macerate at 2500 rpm
for 30 seconds. Repeat by dropping back into liquid Nitrogen and macerating an
additional 2 times. One final cooling in the Nitrogen and you are ready to begin
the DNA extraction.
DNA extraction:
5. Add 1.4 mL Buffer ASL to sample. Careful, the tube will be nearly full. Close
top carefully. Vortex continuously for 1 min or until thoroughly mixed (this
ensures maximum DNA isolation).
6. Heat for 5 min at 70º C.
7. Vortex 15 seconds. Centrifuge at full speed for 2 minutes to pellet debris.
8. Transfer 0.5 mL of the supernatant into each of 2 new 1.5 mL microcentrifuge
tubes. Keep the screw cap tube with debris. The glass beads can be reused up to
five times with sterilization.
9. Insert half of an InhibitEX tablet into each sample and vortex immediately and
continuously for 1 min. Incubate a minimum of 1 min on bench at room
temperature to allow adsorption of inhibitors.
10. Centrifuge at full speed for 10 minutes (we’ll do this as a class- all 4 samples at
the same time).
11. Transfer all the supernatant carefully into a fresh 1.5 mL tube; discard pellet.
Centrifuge at full speed 5 minutes.
12. Add 15 µL Proteinase K into a new 1.5 mL microcentrifuge tube.
13. Transfer 200 µL of supernatant into the tube containing the proteinase.
14. Add 200 µL Buffer AL and vortex 15 s. Be sure all is thoroughly mixed.
15. Incubate at 70º C for 10 minutes.
16. To same tube add 200 µL ethanol (96-100%, room temp) and mix by vortexing
lightly.
17. Carefully apply the complete lysate to a QIAamp spin column with its collection
tube without moistening the rim. Close cap and centrifuge full speed for 2 min.
Place the column into a new collection tube and discard the filtrate.
18. Carefully open spin column and add 500 µL Buffer AW1 (wash 1). Spin at full
speed for 2 min. Again transfer column to new collection tube and discard filtrate.
19. Carefully add 500 µL Buffer AW2 (wash 2). Spin 5 min at full speed.
20. Note color of column. If dark, perform an additional wash with AW2. If still
dark, see me about steps to remedy.
21. Transfer spin column to a labeled 1.5 mL microcentrifuge tube being careful not
to get any filtrate on the bottom tip of the column.
22. Pipet 100 µL Buffer AE (elution) directly onto the QIAamp membrane (not sides
of spin column). Incubate at room temp one minute and then centrifuge at full
speed for 2 minutes to elute the DNA. Place tube containing DNA in refrigerator
until ready to use tomorrow.
Document Control: February 17, 2016