Time: about 5-6 hours, Yield- 400 ug /gr of wet fungal tissue
· Use cut tips for pipetting (not necessary for C. graminicola, but some fungi create a viscous supernatant for which a cut pipette will help)
· All centrifugation steps will be at 4,000 rpm at room temperature (~20˚C)
1. Place 1.4-1.6g of fungal tissue (wet weight) in a 15 mL tube.
2. Lyophilize the tissue for 24 hrs.
3. Break the tissue using a clean glass rod. Be careful to not spill or contaminate the sample.
· Adding 4 glass beads to the tube and vortexing for about a minute may help in breaking up the tissue.
4. Add 5mL of extraction buffer [50mM Tris + 50mM EDTA + 2% SDS, pH=8.0] to each tube and mix for 10 seconds.
5. Incubate tubes for 30 minutes in a 68˚C bath.
6. Centrifuge the tubes for 20 minutes.
6a. Transfer the supernatant to a new tube. Add 300ul of 0.3 M ACK and gently mix by inversion.
Place on ice 10 minutes, then centrifuge for 10 minutes.
Note: this step is recommended to reduce the amount of foam on the supernatant but is not required for successful extraction.
7. Transfer supernatant to a new tube.
8. Add 5.2 mL of phenol-chloroform and mix two times for 8 seconds with a 1 minute interval.
9. Centrifuge for 15 minutes.
10. Transfer supernatant to a new tube and record volume.
The supernatant in this step can sometimes end up inverted due to high salts in the solution. The part you want to keep and take to the next step is the light yellow looking portion.
11. Add that volume in a 1:1 chloroform:isoamylalcohol solution.
For example if the volume of supernatant collected is 5mL add 2.5mL chloroform and 2.5mL isoamylalcohol
12. Centrifuge for 15 minutes.
13. Transfer supernatant to a new tube and record volume.
14. Add an equal volume of isopropanol to the supernatant and mix by inverting.
(This can be used as an over night step if necessary as long as the isopropanol has already been added to the tubes and the tubes are kept in a -20˚C freezer)
15. Centrifuge the tubes for 15 minutes.
16. Discard isopropanol and invert tube to dry pellet. This usually takes several hours.
(The tubes may be left to dry overnight if needed.)
17. Resuspend the pellet in 500µL of RNA free H2O and place the tubes in 68˚C water bath for 5 minutes or 37˚C for 30 minutes.
18. Add 5 µL of 10mg/mL RNase per 500 µL of DNA and incubate the tubes in a 37˚C water bath for 30-60 minutes. (RNase in Fridge #1, 4˚C in a box)
19. Add 1:10 volume of 3M NaOAc (pH 5.2) and 2.5 volume of 95% EtOH to each sample. Mix gently and incubate tubes in -20˚C freezer for at least 20 minutes.
20. Centrifuge the tubes for 10 minutes.
21. Wash twice with 500 µl of 70% ethanol. Centrifuge for 5 minutes for each wash.
22. Invert tubes to dry pellet. This may take several hours. Resuspend DNA in 300µL of RNA free H2O.
Protocol adapted from Dr. Kennerley Lab
Extraction buffer
(50mM Tris + 50mM EDTA + 2% SDS, pH=8.0])
To prepare a 100 ml of extraction buffer
20 ml SDS 10%
5 ml Tris, 1 M
10 ml EDTA, 0.5 M
65 ml of ddH20 (sterilized)
Trouble shooting tips:
1. After step 9, it is very hard to collect the supernatant without disturbing the bottom layer. Collect as much as you can; recentrifuge if necessary.
2. Drying the pellet is usually an overnight step. Simply resuspend the next morning and place on ice if not using right away.
3. You will use 3 additional tubes per sample. It is best to label both the tubes and tops, and also write down sample names, as they can wash off in the water baths.
Last updated on June, 2006