Editing Plant Genomic DNA Isolation Protocols (section)

== DNA extraction protocol  ==

A. Breaking the Plant Cell Wall and Cell Lysis

(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.) <br> <br> 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. <br> <br> 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.) <br> <br> 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.)

4. Spin Tube down at 1,300 X G for 2 minutes to get rid of sand and debree. Transfer the supernatent to a new 50 ml tube and discard sand and debree into trash. 

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. <br> <br> 6. Mix by inverting tube several times. Make sure that the lid is on tight.<br> <br> 7. Centrifuge at 10,000xG (or max on other centrifuge) for 2-3 minutes. <br> <br> 8. Check the color of the aqueous layer.<br> <br> 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. <br> <br> 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). <br> <br> 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. <br> <br> 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. <br> <br> 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.<br> <br> 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 <br> <br> 14. Dump the supernatant, then wash the pellet with about 400 ul of 70% Ethanol. This will get rid of any excess salt. <br> <br> 15. Centrifuge for 5 minutes at maximum speed. <br> <br> 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. <br> <br> 16. Resuspend the DNA in about 700 ul of TE buffer. <br> <br> 17. Rehydrate DNA by incubating sample overnight at room temperature. The sample should be placed in the refrigerator the next day. <br>

<br>

'''Another protocol'''

1. Preheat the extraction buffer containing 100 mM Tris–HCl (pH 8), 1.4 M NaCl, 20 mM EDTA (pH 8), 2% (w/v) CTAB in water bath at 60°C for about 15 minutes.<br>2. Submerge 1 g of plant tissue in 5 ml of absolute alcohol for 5 minutes and allow alcohol to evaporate.<br>3. Grind the tissue in presence of 1% PVP (Polyvinylpyrrolidone) and pre-warmed extraction buffer by using a pre-chilled mortar and pestle (−40°C/−80°C) at room temperature.<br>4. Transfer the ground material into 2 ml centrifuge tubes and incubate in water bath at 60°C for 1 hour.<br>5. Centrifuge the tubes at 10,000 rpm for 10 minutes at 4°C and collect the supernatant in 1.5 ml centrifuge tube using wide bored tip.<br>6. To the supernatant add equal volume of chloroform:isoamyl alcohol (24:1) and mix by inversion for 15 minutes.<br>7. Centrifuge the tubes at 10,000 rpm for 10 minutes at 4°C and collect the supernatant in 1.5 ml centrifuge tube.<br>8. Again add equal volume of chloroform: isoamyl alcohol (24:1) to the supernatant and mix by inversion for 15 minutes.<br>9. Centrifuge the tubes at 10,000 rpm for 10 minutes at 4°C and collect the supernatant.<br> 

10. To the supernatant add twice the volume of chilled isopropanol to precipitate the DNA and incubate it at −20°C for 30 minutes.<br>11. Centrifuge the tubes at 10,000 rpm for 10 minutes at 4°C and collect the pellet.<br>12. Wash the pellet 2 –3 times with 70% ethanol and air dry the pellet in room temperature.<br>13. Add 50–100μl of TE buffer to dissolve the DNA.<br>14. Store at −20°C for further use<br> 

=== Reagents  ===

100 mM Tris-HCl (pH 8) 

1.4 M Sodium Chloride (NaCl) 

20 mM-Ethylenediaminetetraacetic acid (EDTA) (pH 8)<br>2% (w/v) Cetyl trimethylammonium bromide (CTAB)<br>Chloroform-isoamyl alcohol (24:1)<br>Isopropanol 

70% ethanol<br>TE buffer (pH 8): 10 mM Tris–HCl, 1 mM EDTA<br>