Tim Chipman (July, 1995)

I have attempted a number of total nucleic acid preps using C. richardii prothalli and have had some degree of success. It appears that the limiting factor in this protocol is the amount of tissue readily available. I have been culturing 2.5L batches of spores according to the C-Fern Manual (250mg of spores in 2.5L of growth medium). Batches of this size yield approximately 5 grams of tissue upon harvesting. In all cases, prothalli were harvested after 2 weeks of growth, by straining through a cheesecloth stretched over a funnel mouth. It is likely that when more lab space is available here, I will attempt to grow mature sporophytes and then attempt DNA preps using larger amounts of tissue harvested from the sporophytes.

I attempted the following basic methods of nucleic acid isolation:

1. Subcellular Fractionation Protocol (Spencer, D.F., Gray, M.W.and Schnare, M.N. The isolation of mitochondrial DNA and RNA. Modern Methods of Plant Analysis: New series vol.14. Seed Analysis. Ed: Linskens, H.F. and Jackson, J.F. pg. 348-360)

   In brief, this protocol involves: Grinding tissue in Tris/EDTA buffer, followed by a number of centrifugation steps (900g for 6 minutes, 1900g for 6 minutes, 17000g for 20 minutes) to separate the various fractions (crude nucleic, cytosolic RNA, mitochondrial). Nucleic acid and cytosolic RNA yield were reasonable, however, mtDNA yield was very low. DNA was purified using standard phenol extraction / ethanol precipitation techniques.

2. Total Nucleic Acid Extraction:

   Grind tissue in 1:1 mix of buffer solution (0.5M Tris / 0.2M EDTA / 2% Sarkosyl / 0.3M sodium acetate) and pH 8.0 Tris-equilibrated Phenol. Crude spin with centrifuge (5 minutes @ 10,000 RPM), followed by phenol extractions of aqueous layer for cleanup. Ethanol precipitation, later followed by additional phenol extractions to cleanup sample. Yield from this method was very poor, possibly due to inadequate grinding of cells at the initial stages.

3. Liquid N2 / Hot CTAB Extraction:

   Squeeze tissue dry in cheesecloth, transfer to thermos of liquid nitrogen to flash-freeze. Transfer frozen tissue to pre-cooled mortar & pestle, grind on liquid nitrogen to yield a fine powder. Transfer powdered tissue to hot (65 deg.C) CTAB buffer (2% CTAB / 1% PVP / 1.4M NaCl / 20mM EDTA / 100mM Tris-HCl), invert to mix. Leave at 65 deg. C for 15 minutes, mixing periodically. Allow to cool to room temperature, then cleanup with 2 reps of 1 vol isoamyl:Chloroform (24:1). Precipitate nucleic acid with isopropanol, cool to 4 degrees C. Spin down nucleic acid gently, dump supernatant, rinse, dry, add sodium acetate and phenol extract 3x to cleanup. Store at -20 deg.C in EtOH.

   This third method had a moderate yield, but there was something which extracted with the nucleic acid - likely carbohydrate - which made life difficult.

4. Liquid N2 / Guanidinium Isothiocyanate Extraction:

   Essentially as per method 3, but using a 6M guanidinium isothiocyanate solution in place of the CTAB buffer. DNA yield was abysmal.

The descriptions here are rather brief, but hopefully they will help someone out a bit. If anyone wants any more information on these protocols, feel free to contact me at: tchipman@is.dal.ca