Biology 475 Molecular Biology Genomic DNA Isolation from Microbial Mats Sarah Boomer, copyright 2003

National Science Foundation Western Oregon University Yellowstone National Park

Methods for isolating genomic DNA vary as a function of sample origin and chemistry.  You should thoroughly consult the literature in your particular field for appropriate methods. This protocol is widely applicable and serves to isolate genomic DNA from many bacteria and animal cells.   Our samples, unstudied bacterial mat communities dominated by red filamentous microorganisms, were collected in Yellowstone and frozen at minus 65 degrees Celsius.   They are irreplaceable - BE CAREFUL. 

Following the procedures below, you will lyse, stabilize, and clean your samples using the following strategies and compounds: EDTA stabilizes DNA and inhibits released cellular nucleases.   Lysozyme digests the bacteria cell wall. SDS, a detergent, gently disrupts the cell membrane. Phenol/chloroform, an organic solvent that forms a distinct lower layer when mixed with aqueous liquids, extracts most cell parts (including most proteins, membranes and pigments), leaving primarily DNA in the upper layer.  Saving only the latter, we selectively precipitate the DNA by adding salt and alcohol. 

The destination of your genomic DNA will be PCR amplification and cloning.  Because PCR can detect as few as one target sequence, it is IMPERATIVE that all equipment and reagents used in the genomic isolation be dedicated to genomic/PCR work only.  We use segregated ultra-pure chemicals, bottled water, centrifuge tubes, and plugged tips.  We carry out these procedures in ultra-clean workspaces using UV-sterilized pipettemen.  People who carry out these procedures should do so only if they have handled no DNA whatsoever that day.  Following these procedures, space and equipment is bleached and treated with a hand-held UV.

Procedures

Obtain mat sample in 300 ul fresh GTE buffer.

Add 40 ul lysozyme (frozen stocks, 10 ug/ml).

Add 75 ul EDTA (0.5 M, pH 8) and incubate at 37°C for 30 minutes.

Add 50 ul 10% SDS and invert several times.

IN THE FUME HOOD AND WEARING GLOVES:  Perform TWO extractions using phenol/chloroform.  The phenol/chloroform is the LOWER layer, beneath the aqueous layer of TE buffer.  This means you have to dip down to the lower layer to get the phenol/chloroform from the reagent bottle.

Add 500 ul phenol/chloroform (lower layer of reagent bottle)
Vortex one minute (hold upright and carefully to avoid leakage)
Centrifuge at max speed (14K rpm) for 5 minutes - room temp.
Transfer aqueous layer to new tube;  withdraw SLOWLY from the top
It is better to leave behind crud if visible at interface
Repeat procedure one more time

Make sure ALL phenol waste is placed in NS201 hood and labeled "phenol waste."

Precipitate DNA by adding sodium acetate (3M) to a final concentration of 0.3 M. Unless you have experienced great product loss, this will be 50 ul. Invert immediately after adding salt.

Add two volumes 95% cold ethanol.  Invert/shake and observe for filaments of ACTUAL DNA. Chill 20 minutes in minus 65 freezer.

Centrifuge cold at max speed (14K rpm) for 15 minutes

Carefully decant and record appearance of pellet. 

Wash with 70% COLD ethanol one time. You can do this by carefully pipetting 500 ul onto the pellet and decanting.  As you add the ethanol, make sure the pellet does not dislodge. If the pellet dislodges, centrifuge 5 minutes before decanting.  Record final appearance of the pellet. Show me the pellet.

NOTE:  When you decant the final wash, DO NOT turn tube right-side-up.  Carry it upside-down to wherever you are going to dry it.  This keeps wash from falling back down to the bottom of the tube and onto your drying pellet.

Dry open and upside-down in the fume hood (will take 10-15 minutes). If you are feeling really risky, use a CLEAN kimwipe tissue to wipe down sides of the tube while inverted.  Alternatively, ask me to do this.

I will advise how much water (30-100 ul) to add to your pellet as it takes experience to make this judgment. Gently pipette up and down to dislodge the pellet as you add the water.  Incubate at 37°C for 10 minutes.

Perform PCR IMMEDIATELY; degradation begins immediately and often destroys samples within 1-3 weeks.  After PCR is complete, run remaining genomic DNA sample on a gel alongside PCR product.