Dirty Genomic DNA Extraction
This protocol works well for Mimulus leaves, but may need to be optimized for other tissues or species. It is a very dirty extraction method that works well for PCR. Sometimes, DNA will need to be diluted before PCR. Yield will be low (do not try to quantify DNA), but it works well for PCR.
1) Harvest 1 small, newly expanding leaf from each sample. Leaf size should have an area about 1-2 cm2. Place in the bottom of 1.5 mL tube. For short term, store sample on ice until ready to grind. Otherwise store in -80 freezer.
2) Pour liquid nitrogen into a ceramic mortar. (Liquid nitrogen is on 2nd floor of Bio-Sci). Submerge the outside of the 1.5 ml tube in the liquid nitrogen (while holding the lid), along with a blue pestle for grinding. Once both are cold, grind the leaf tissue to a fine powder (only takes a few strokes).
3) Add 400 uL of DNA extraction buffer (recipe is below) and vortex vigorously for 10 sec. Leave the tubes at room temperature while processing additional samples.
4) Centrifuge at 13,000 RPM for 4 minutes at room temperature.
5) Carefully remove tubes, being sure not to disturb the pelleted plant tissue.
6) Pipet 300 uL of supernatant into new tube (be careful not suck up any plant tissue from the pellet. If the pellet is disturbed, you can re-spin for 2 min).
7) Add an equal volume (300 ul) of isopropanol (2-propanol) and mix the tubes by inverting 4-6 times. Precipiate DNA at room temperature for 5 minutes.
8) Centrifuge at 13000 RPM for 5 minutes at room temperature.
9) Remove the supernatant, keeping in mind the location of the DNA pellet (you will probably not be able to see it). It's OK to leave some isopropanol in the tube - it's better not to disturb the pellet.
10) Wash with 500 ul of 70% ethanol.
11) Spin at 13000 RPM for 3 minutes.
12) Remove the ethanol and pat the mouth of the tube on a kimwipe. Let the pellet dry at room temperature (to speed up the drying process, you can also place the tubes with open lids in the hybridization oven at around 45 degrees, with the door cracked open slightly). This should take about 10 minutes. Make sure there is no more ethanol in the tube, as this will affect PCR.
13) Resuspend the pellet in 100 ul of TE (pH 8.0) or water. Don't pipet up and down vigorously. Leave the tubes at room temperature for 30 minutes, and then they can be used for PCR (1 ul per reaction) or stored at -20 C.
200 mM Tris HCl pH 7.5
250 mM NaCl
25 mM EDTA
0.5 % SDS