Genomic DNA Extraction

Ben Carter, 9/24/2014

Tissue Homogenization

 * 1) Prepare a labeled microfuge tube containing 500µL of 2x CTAB for each sample.
 * 2) Harvest a rosette leaf, 2-3 cauline leaves, or 3-5 seven-day-old seedlings from each plant line to be genotyped. Immediately place tissue in 2X CTAB and grind using a blue pestil until there are no visible green chunks (about 1 minute). Vortex to mix. Samples can sit in the 2x CTAB while others are being harvested.
 * 3) Incubate tubes on a 65°C heat block for 1h.

Organic Extraction
Note: Add the included Tris buffer to the PCI solution, invert 5x, and let sit for 2-4h before first use.
 * 1) Add 500µL of PCI (Thermo-Fisher Cat# BP17521 400) to each tube in a fume hood. Vortex vigorously for 1min.
 * 2) Centrifuge at max speed in a room temperature microfuge for 5min.
 * 3) Transfer the aqueous layer (top, clear layer) to a new labeled microfuge tube. Do not transfer any of the organic phase or interface: purity over quantity!

Nucleic Acid Precipitation

 * 1) Add 2/3 volumes of isopropanol to the tube. Invert 5x to mix. Incubate at room temperature for 10min. If you need to stop here, use 3 volumes of ethanol instead of isopropanol and store at -20°C overnight.
 * 2) Centrifuge at max speed in a room temperature microfuge for 15min to collect the precipitated DNA.
 * 3) Gently pipette away the supernatant. Gently add 300µL of freshly prepared 70% ethanol. Repeat centrifugation for 5min.
 * 4) Gently pipette away the supernatant. Spin on a tabletop microfuge and remove any residual supernatant.
 * 5) Air dry the pellet by placing the open microfuge tubes in the laminar flow hood for 10min.
 * 6) Dissolve the pellet in 50µL of nuclease-free water or TE Buffer. Ensure the pellet is dissolved by inspecting the tube for Schlieren lines. Schlieren lines are where light is distorted from a solvent-pellet surface.
 * 7) Store DNA samples at -20°C

RNAse Treatment (optional)
''Note: this step is not required for routine PCR. It is beneficial when performing Illumina sequencing.''
 * 1) Add 2µL of 10mg/mL RNAse A solution to the tube. Pipette to mix. Incubate at room temperature for 30min.
 * 2) Perform the steps detailed under the Organic Extraction and Nucleic Acid Precipitation headings above to remove the RNAse enzyme from the samples. One modification: add 1/10 volume of 3M sodium acetate pH 5.2 to the sample along with the isopropanol.

2x CTAB

 * 1g of CTAB (cetyltrimethyl ammonium bromide)
 * 4.09g of sodium chloride
 * 5mL of 1M Tris-Cl pH 8.0
 * 2mL of 0.5M EDTA pH 8.0
 * Dilute to 50mL with Milli-Q water.

TE Buffer

 * 5mL of 1M Tris pH 8.0
 * 1mL of 0.5M EDTA pH 8.0
 * Dilute to 500mL using Milli-Q water
 * Filter sterilize