DNA/RNA extraction

DSCN3179

Genomic DNA extraction using Chloroform-CTAB

Grow mycelium in liquid culture for 2-5 days depending from the growth speed.

Harvest mycelial biomass using Miracloth, filter out the medium, wash out several times with sterile ddH­2O.

Cool down mortar and pestle with liquid nitrogen, put mycelium there and sprinkle it with sterile acid washed sand.

Grind mycelium very thoroughly (not less than several hundred pestle moves), keeping mycelium frozen the entire time with liquid nitrogen.

Prepare 1.7-2 ml microcentrifuge tubes with 500 ul CTAB, in fume hood add 5 ul β-mercaptoethanol and 5 ul of 20 mg/ml Proteinase K to each tube.

Put ca. 100 mg mycelium with sterile spatula in each tube, vortex, no clumps.

Incubate the tubes into water bath 65°C for 30-60 min mixing at least once (better to skip this step for many fungi because of fast DNA degradation).

In fume hood add 500 ul Chloroform:Isoamylalcohol 24:1, and mix by gently shaking tubes.

Centrifuge at 14 000 rpm for 10 min.

Carefully (avoid any Chloroform*) transfer the upper phase, pipetting it into a new labelled microcentrifuge tube with cold 500 ul Isopropanol*, slowly invert watching DNA aggregation and enjoy it.

If precipitated DNA builds visible threads, use sterile glass hook to spool them and transfer into another microcentrifuge tube with a little sterile ddH­2O.

If no visible DNA threads, pellet it using centrifugation at 14 000 rpm for 10 min.

Discard supernatant, watch the pellet. Invert tubes on a clean paper towel and allow pellets drying for 10-15 minutes upside down, or until pellets looks dry.

Wash pellet one-two times with cold 70-80% Ethanol.

Centrifuge at 14 000 rpm for 1 min.

Discard Ethanol, watch the pellet.

Dry out pellet on clean tissue inverting microcentrifuge tube as above.

Hydrate pellets with 25-50 ul sterile ddH­2O.  If needed use water bath (see above) to warm up the tubes and dissolve the pellet pipetting up and down several times.

Add 1.0-1.5 uL RNAse A.  Incubate at 37°C for 15 minutes.

Check 1 ul of DNA. For that use gel, NanoDrop and Qubit.

Use 0.8% agarose gel for quality estimation: size of HMW DNA, degradation smear and presence of RNA bands, and roughly for quantity comparing to ladder bands intensity.
Use NanoDrop to estimate DNA contamination using absorption ratios at 260/230 (impurity with carbohydrates, peptides, phenols) and 260/280 (proteins, aromatic amino acids), and roughly quantity).
Use Qubit for precise DNA quantity evaluation.

Store the sample at -80°C.

Labeling is very important!!!

*Chloroform and Isopropanol wastes should be disposed of separately in a glass bottles with a chemical waste labels.

CTAB Buffer
100 ml 1 M Tris HCl pH 8.0
280 ml 5 M NaCl
40 ml  of 0.5 M EDTA
20 g of CTAB (cetyltrimethyl ammonium bromide)
Bring total volume to 1 L with ddH­2O.

Similar technique: Filamentous fungi genomic DNA isolation protocol

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