This protocol uses the Nucleon ® DNA Extraction Kit from Amersham Life Sciences ® and will enable you to purify approximately 10 - 50 ug of DNA. This DNA will be used subsequently for Southern Blotting Analysis. Each group will isolate genomic DNA from two sources, one source will be Arabidopsis thaliana, and the second will be another common plant in the same family (the Brassicacae Family) such as broccoli or cauliflower.
I. Breaking the Plant Cell Wall
(The purpose of this step is to break down the cell wall of the plant. It is thus important to get the plant tissue very well ground up, but this procedure needs to be done quickly to prevent your DNA from degrading during the procedure.)
1. In mortar add 1 g of wet plant tissue and 4.6ml of reagent 1. Grind tissue using pestule to yield a fine powder. Pour material into a 15ml microfuge tube. Use gloved finger to transfer as much material as possible. Clean mortar and pestule and repeat with 1 g of second plant. Transfer into a different 15ml microfuge tube. Make sure to label both 15ml tubes. Keep the tubes on ice to minimize DNase activity
II. Cell Lysis
2. To each tube add 23 ul of RNase mix. This solution should be incubated at 37 degrees Celcius for 30 min.
3. After incubation, add 1.5 ml of Reagent 2 and invert until a homogenous mixture is obtained. Incubate in a water bath at 65 degrees Celcius for 10 min. During this incubation time the tube should be frequently inverted to keep the contents well mixed.
4. Place the samples on ice for 20 min.
(The Active ingrediants in this process is the detergent, SDS.)
III. DNA Extraction with Nucleon PhytoPure resin and chloroform
(Two important chemicals are used in this step Chloroform and the Nucleon Phytopure resin. Each chemical is used to help seperate proteins and polysacharides from nuceic acids in the cell. The purpose of the Chlorform will be to help bind up the complexed proteins and polysaccharides. Chloroform is more dense than water solutions and thus after spinning this solution Chloroform and water will seperate into two distinct phases. The lower phase will be Chloroform. This is the phase that proteins and polysacharides find most chemically attractive. The upper aqueous phase will contain your DNA. With plant tissue there is often more polysacharides than in animal tissue. If the polysacharrides are not removed many procedures such as restriction enzyme digests will not work appropriatly. The Nucleon Phypure DNA extraction resin like Chlorform is attracted to polysacharides and will help ensure that none remain in the aqueous layer. This resin will form a brown layer in between the chloroform and the aqueous phases of your solution.)
5. After 20 minutes, remove the sample from ice and add 2 ml of chloroform that is chilled to -20 degrees Celcius. This step should be done in the fume hood.
6. Add 200 ul of Nucleon PhytoPure DNA extraction resin suspension. (Note: Ensure that the resin is fully suspended by vigorous shaking immediatly before use. It is also important to ensure that the resin bottle contains equal proportions of resin and buffer. If this is not the case add a volume of TE buffer to the resin to make the proportions equal.)
7. Incubate this mix for 10 minutes at room temperature, frequently inverting the tubes to mix.
8. Centrifuge at 1,300xG for 10 minutes.
9. Without disturbing the lower brown layer of resin, transfer the upper greenish colored liquid into a fresh 15 ml tube. (If you feel you did disturb the brown layer of resin in your transfer- try doing an additional spin at a higher speed to clarify the transferred aqueous phase. Transferring again the top of this tube into a new one.)
IV. DNA Precipitation
10. Add an equal volume of ice cold isopropanol to your sample. Mix the tube gently by inversion until DNA parcipitates (5-10 min.)
11. Centrifuge the tube at the maximum speed(3,780 RPM on floor centrifuge) for ten minutes to pellet the DNA.
12. Discard the supernatent and add cold 70% ethanol to the pellet. Mix by inversion.
13. Centrifuge the tube for an additional five minutes to repellet the DNA.
14. Discard the supernatent and allow the pellet to air dry until all traces of ethanol are removed.
15. Resuspend the DNA in about 400 ul of TE buffer.
16. Rehydrate DNA by incubating sample 1 hour at 65 degrees Celcius or overnight at room temperature.
17. The concentration of the DNA will be determined using the UV-
spectrophotometer. A demonstration of how this is done will given in
class, but before measuring you sample work through the following
tutorial.
The concentration of DNA is read by measuring the absorbence of a sample at A260 on a spectrophotometer. The DNA sample is placed in a quartz cuvette in order to read the optical density (OD). Common glass and plastic will also absorb at this wavelength so special quartz cuvettes that do not absorb light at this wavelength are used to measure the absorbence of the DNA. Be very careful with these, as they are very expensive.
Scientists have determined an absorbence coefficient to use to determine the concentration of DNA in your sample preparation. The A260 of DNA with the concentration of 50 mg/ml is 1 OD unit. To be accurate your A260 readings should be between 0.1 and 1.
Let's say your DNA sample was diluted 2 ml into 1000 ml and this dilution had an A260 of 0.25. How would you determine the concentration of your original DNA solution?
The concentration of DNA in the original solution would be 5 mg/ml.
Scientists often measure the absorbence of their DNA sample at A280 as well as A260. Both proteins and nucleic acids absorb light at 280 nm. By obtaining the ratio of A260/A280 scientists can thus get an idea of the purity of their DNA sample. For good quality DNA without a lot of protein this ratio should be between 1.8 and 2.
Before measuring your concentration you will need to prepare two blank samples (water) to zero the machine (1 ml each), and a dilution of each of your DNA samples in water.
If we estimate that we isolated between 25- 100 mg of DNA, how would you dilute your DNA to insure that you have a reading between 0.1 and 1, knowing that you need 1 ml of diluted DNA for a reading? Note you want to use as little of you stock DNA as possible but you need to use enough of it to pipet accurately