Step 1: Orchid Total DNA Extraction (DNeasy Plant Mini Kit – Qiagen)
1. Set volumes on two pipettors: 400 ul and 4 ul. Obtain gloves.
2. A. Record species name (check spelling!)
B. Obtain and label a microcentrifuge tube.
C. Carefully remove a small amount (< 100 mg (0.1 g)) of tissue (leaf tip on a younger leaf) using a fresh razor blade.
D. Finely dice tissue.
E. Obtain mortar/pestle from freezer, place tissue in mortar, carefully cover with liquid nitrogen.
F. Start by slowly crushing tissue until liquid nitrogen has almost evaporated.
G. Crush tissue by rotating pestle… grind tissue into a uniform fine powder.
H. Using spatula, scrape powdered tissue into tube as quickly as possible.
Note: Work quickly to avoid thawing of tissue. There is less chance of DNase damage if tissue remains frozen.
3. A. Add 400 ul of AP1 and 4 ul of RNase A to sample.
B. Vortex to mix (no tissue clumps should remain) and incubate at 65 C for 10 min.
C. Invert 2-3 X during incubation (IMPORTANT!)
This step lyses the cells and removes RNA.
4. Add 130 ul of AP2, mix and incubate on ice for 5 min.
This step precipitates detergent, proteins and polysaccharides.
5. Centrifuge to pellet all insoluble materials at 20,000 x g for 5 min.
This step minimizes DNA shearing that can result if lysate is viscous (and passes through spin column – next step). DNA is in the supernatant.
6. A. Transfer supernatant into QiaShredder Mini spin column in a 2 ml collection tube.
[Keep carryover of pellet material to a minimum.]
B. Spin at 20,000 x g for 2 min.
7. Transfer the flow-through into a new tube (set pipettor to 450 ul). If you have significantly more or less supernatant, adjust pipettor until you can get a more accurate estimate of flow through volume.
8. Add 1.5 volumes of AP3 and mix well by inversion.
9. A. Place a DNeasy Mini column into a 2 ml collection tube.
B. Pipet 650 ul of supernatant-AP3 mixture into Mini column
C. Spin at 6000 x g for 1 min.
D. Discard flow-through
E. Repeat 9B-D with remaining mixture and discard the collection tube (NOT the column)
This step facilitates binding of the DNA to the DNeasy minicolumn filter
10. A. Place the DNeasy minicolumn in a new 2 ml collection tube.
B. Add 500 ul Buffer AW
C. Spin for at 6000 x g for 1 min.
D. Discard flow-through (keep the collection tube!)
11. A. Add 500 ul Buffer AW to the mini column
B. Spin at 20000 x g for 2 min.
These steps (#10 and 11) “wash” the DNA to remove salts. The higher speed spin assures removal of residual alcohol from DNA and dries it.
12. A. Remove the minicolumn to a 1.5 ml centrifuge tube. (avoid any carryover of flow-through!)
B. Add 100 ul of Buffer AE onto the minicolumn membrane (Do NOT touch membrane with pipet tip!)
C. Incubate at room temperature for 5 min.
D. Spin at 6000 x g for 1 min.
13. Repeat 12B-D and discard minicolumn
These steps (#12 and 13) elute the DNA from the column. The DNA is now highly purified. We will check quality via gel electrophoresis and quantify the DNA using a NanoDrop Spectrophotometer. If sufficient yield and purity, it can be used for a variety of downstream applications.
Step 2: Use 5 ul of extraction for gel electrophoresis. Only 1 ul is required for quantification (use Buffer AE as the blank!!).
DNA largely in the form of high molecular weight fragments is most desirable. This will appear as a bright large band with a minimal “smear” of smaller fragments. While PCR is very robust even when DNA is not in this optimal state, high molecular weight DNA is indicative of good extraction technique (and an extraction kit that is working well).
With this kit, yield of 6 – over 20 ng/ul are typical. I have found that 10 ng of total DNA is sufficient for amplification of plastid targets. To simplify set up of PCR reactions, aliquots of extracted DNAs will be diluted to 1 ng/ul. 10 ul of these dilutions is then used in each PCR reaction.
ArmstrongGenetics3 