(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. Cut frozen plant tissue into pieces as fine as possible with a razor blade. In mortar, add 4-10 leaves of cut up plant tissue and 5 ml of CTAB reagent (100mM Tris pH= 8.0, 1.4M NaCl, 25 mM EDTA, 2% CTAB, 0.2% beta-mercaptoethanol). Grind tissue using pestle to yield a fine slurry.

 

2. Add 15 ml more CTAB buffer to the mortar and continue to grind until of uniform texture. Pour material into a 50ml centrifuge tube. Use a rubber policeman to transfer as much material as possible. Clean the mortar and pestule and repeat with second plant. Transfer the next slurry into a different 50ml microfuge tube. Continue to repeat procedure until all samples are ground up. (Make sure you label all 50ml tubes.)

 

3. Place the tubes in a 50 C water bath for approximately 1 hour. (The Active ingredient in this process is the detergent, CTAB. EDTA has been added to deter Dnase activity.)

B. DNA Extraction: chloroform/isoamyl alcohol (or octanol) (24:1) Two important chemicals are used in this step, Chloroform and alcohol. Each chemical is used to help separate proteins and polysaccharides from nucleic acids in the cell. Chloroform is more dense than water solutions, thus after spinning this solution, the chloroform and water will separate into two distinct phases. The lower phase will be Chloroform/alcohol. This is the phase that proteins and polysaccharides find most chemically attractive. The upper aqueous phase will contain your DNA. With plant tissue there are often more polysaccharides than in animal tissue. If the polysaccharides are not removed, many enzymatic procedures such as PCR or restriction enzyme digests will not work appropriately. These steps should be performed in the fume hood.

5. Add 25 ml of chloroform/octanol.

 

6. Mix by inverting tube several times. Make sure that the lid is on tight.

 

7. Centrifuge at 10,000xG (or max on other centrifuge) for 2-3 minutes.

 

8. Check the color of the aqueous layer.

 

If it is clear= Without disturbing the lower green level (chloroform) or the middle whitish layer (various organics and parts of leftover plant tissue), transfer the upper clear colored liquid into a fresh 50 ml tube and continue onto the steps below. Follow your instructors guide in disposing of chloroform properly.

 

If it is cloudy= Without disturbing the lower green level (chloroform) or the middle whitish layer (various organics and parts of leftover plant tissue), transfer the upper cloudy colored liquid into a fresh 50 ml tube, add more CTAB to a volume of 20 ml and then redo steps 5, 6,7 and 8. Follow your instructors guide in disposing of chloroform properly

C. DNA Precipitation

9. Add an equal volume (approx 25ml) of ice cold isopropanol to your sample. Mix the tube gently by inversion until DNA precipitates (5-10 min.) Place on ice for 10 minutes. (if you have a lot = pull out the pellet of DNA and place it in a 2 ml tube and go to step 11a, if you don't have a visible pellet go to step 10).

 

10. Centrifuge the tube at 10,000 X G for six minutes (or two times 3 minutes if you need to work around students who are still trying to get clear aqueous layers) to pellet the DNA. Now go to step 11b.

 

11a. Dissolve pellet in approximately 1000ul of TE. Mix by inversion and place in the 50 degree incubator until completely dissolved or you have tried to dissolve it for at least 10-15 minutes. Transfer 500 ul of solution to a new 2 ml microfuge tube and proceed to step 12.

 

11b. Dump supernatant. Dissolve pellet in approximately 500ul of TE. Mix by inversion and place in the 50 degree incubutar until completely dissolved or you have tried to dissolve the pellet for at least 10 min. Transfer 500 ul of solution to a 2 ml microfuge tube and proceed to step 12.

 

12. Add 1/10 volume (about 50ul) of 7.5M ammonium acetate or 3M sodium acetate (pH= 5.2) and 2.1 volumes of ethanol (about 1100ul). Mix

13. Centrifuge for 10 minutes in the microfuge at maximum speed

 

14. Dump the supernatant, then wash the pellet with about 400 ul of 70% Ethanol. This will get rid of any excess salt.

 

15. Centrifuge for 5 minutes at maximum speed.

 

15. Discard the supernatant and centrifuge for 30 seconds. Remove all supernatant with a pippette tip and then allow the pellet to air dry until all traces of ethanol are removed.

 

16. Resuspend the DNA in about 700 ul of TE buffer.

 

17. Rehydrate DNA by incubating sample overnight at room temperature. The sample should be placed in the refrigerator the next day.

D. Determination of DNA Concentration - next period- please read and work through the questions before coming to class!!!

The concentration of DNA is found by measuring the absorbance of a sample at 260 nm on a spectrophotometer. The nitrogenous bases absorb light of this wavelenght but unfortunately so does common glass and plastic so special quartz cuvettes that do not absorb light at this wavelength are used to measure the absorbance of the DNA. Thus, you can either use a quartz cuvette and a UV spectrophotometer for this process or a machine called a nanodrop which does not require a cuvette aand requires only a small amount of sample. For this class we are going to use the departmental nanodrop.

You will be placing 2 ul of your sample onto the nanodrop that is calabrated with TE buffer by the class to determine the concentration of DNA in your sample preparation. The A260 or absorbance at 260 nm of DNA with the concentration of 50 ug/ml is 1 opitcal density(O.D.) unit . Thus the machine calculates the concentration of your sample by multipling the absorbance of your sample at A260 by 50 ug/ml. To be accurate though, your final concentration should not be above 3 ug/ul. If it is please dilute your sample and try the nanodrop again. (Note this is just a modification of Beer's Law that you learned in chemistry. Check out Beer's law for a reminder on how this works.)

Scientists often measure the absorbance of their DNA sample at 280 nm as well as at 260 nm. 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.

• Although the machine will calculate this for you - be prepared to calculate it yourself on the lab exam:
• If you had an OD of 0.567 at A260 and an OD of 0.273 at A280, would this sample of DNA be considered pure? Why or why not?
• If you had an OD of 0.567 and had diluted the sample 10 fold before taking the nanodrop reading, what would the final concentration of the sample be?