Science Buddies: "Ask an Expert"

1. Gather healthy green leaves from two medicinal plants.
2. Give each plant a unique identification code.
3. For each sample, pipette 200 μl of sterile double-distilled water (ddH2O) into a PCR tube.
4. Grind 200 mg of plant tissue to a fine paste in approximately 500 μl of CTAB buffer.
5. Transfer CTAB/plant extract mixture to a microfuge tube.
6. Incubate the CTAB/plant extract mixture for about 15 min at 55o C in a recirculating
water bath.
7. fter incubation, spin the CTAB/plant extract mixture at 12000 g for 5 min to spin
down cell debris. Transfer the supernatant to clean microfuge tubes.
8. To each tube add 250 μl of Chloroform : Iso Amyl Alcohol (24:1) and mix the
solution by inversion. After mixing, spin the tubes at 13000 rpm for 1 min.
9. Transfer the upper aqueous phase only (contains the DNA) to a clean microfuge
tube.
10. To each tube add 50 μl of 7.5 M Ammonium Acetate followed by 500 μl of ice cold
absolute ethanol.
11. Invert the tubes slowly several times to precipitate the DNA. Generally the DNA can
be seen to precipitate out of solution. Alternatively the tubes can be placed for 1 hr at
-20 o C after the addition of ethanol to precipitate the DNA.
12. Following precipitation, the DNA can be pipetted off by slowly rotating/spinning a
tip in the cold solution. The precipitated DNA sticks to the pipette and is visible as a
clear thick precipitate. To wash the DNA, transfer the precipitate into a microfuge
tube containing 500 μl of ice cold 70 % ethanol and slowly invert the tube. Repeat.
((alternatively the precipitate can be isolated by spinning the tube at 13000 rpm for a
minute to form a pellet. Remove the supernatant and wash the DNA pellet by adding
two changes of ice cold 70 % ethanol )).
After the wash, spin the DNA into a pellet by centrifuging at 13000 rpm for 1 min.
Remove all the supernatant and allow the DNA pellet to dry (approximately 15 min).
Do not allow the DNA to over dry or it will be hard to re-dissolve.
13. Resuspend the DNA in sterile DNase free water (approximately 50-400 μl H2O; the
amount of water needed to dissolve the DNA can vary, depending on how much is
isolated). RNaseA (10 μg/ml) can be added to the water prior to dissolving the DNA
to remove any RNA in the preparation (10 μl RNaseA in 10ml H2O).
14. After resuspension, the DNA is incubated at 65o C for 20 min to destroy any DNases
that may be present and store at 4o C.
15. Repeat steps for the other two plant leaves. Store the tubes of plant DNA in the refrigerator until we are ready to proceed to the PCR steps.

MY PROGRESS IN PROJECT:
Now i am done placing order for my Primer sequence from a company.
I am done with isolation of the Chloroplast genome. Next week we will start with PCR amplification & Electrophoresis..
So these 3 days i will be studying Why only those particular chemical reagents (as given in SCIENCE BUDDIES) are chosen & not others..

1. Gather healthy green leaves from two medicinal plants.
2. For each sample, pipette 200 μl of sterile double-distilled water (ddH2O) into a PCR tube.
3. Grind 200 mg of plant tissue to a fine paste in approximately 500 μl of CTAB buffer.
4. Transfer CTAB/plant extract mixture to a microfuge tube.
5. Incubate the CTAB/plant extract mixture for about 15 min at 55o C in a recirculating
water bath.
6. After incubation, spin the CTAB/plant extract mixture at 12000 g for 5 min to spin
down cell debris. Transfer the supernatant to clean microfuge tubes.

7. To each tube add 250 μl of Chloroform : Iso Amyl Alcohol (24:1) and mix the
solution by inversion. After mixing, spin the tubes at 13000 rpm for 1 min.
8. Transfer the upper aqueous phase only (contains the DNA) to a clean microfuge
tube.
9. To each tube add 50 μl of 7.5 M Ammonium Acetate followed by 500 μl of ice cold
absolute ethanol.
10. Invert the tubes slowly several times to precipitate the DNA. Generally the DNA can
be seen to precipitate out of solution. Alternatively the tubes can be placed for 1 hr at
-20 o C after the addition of ethanol to precipitate the DNA.
11. Following precipitation, the precipitate can be isolated by spinning the tube at 13000 rpm for a
minute to form a pellet. Remove the supernatant and wash the DNA pellet by adding
two changes of ice cold 70 % ethanol.
After the wash, spin the DNA into a pellet by centrifuging at 13000 rpm for 1 min.
Remove all the supernatant and allow the DNA pellet to dry (approximately 15 min).
Do not allow the DNA to over dry or it will be hard to re-dissolve.
12. Resuspend the DNA in sterile DNase free water (approximately 50-400 μl H2O; the
amount of water needed to dissolve the DNA can vary, depending on how much is
isolated). RNaseA (10 μg/ml) can be added to the water prior to dissolving the DNA
to remove any RNA in the preparation (10 μl RNaseA in 10ml H2O).
13. After resuspension, the DNA is incubated at 65o C for 20 min to destroy any DNases
that may be present and store at 4o C.

DNA quality confirmation
– Prepare a 1 % solution of agarose by melting 1 g of agarose in 100 mL of 0.5x TBE
buffer in a microwave for approximately 2 min. Allow to cool for a couple of
minutes then add 2.5 μl of ethidium bromide, stir to mix.
– Cast a gel using a supplied tray and comb. Allow the gel to set for a minimum of 20
min at room temperature on a flat surface.
– Load the following into separate wells
10 μL 1kb ladder
5 μL sample + 5 μL water + 2 μL 6x Loading Buffer
– Run the gel for 30 min at 100 V
– Expose the gel to UV light and photograph (demonstration)
– Confirm DNA quality, presence of a highly resolved high molecular weight band
indicates good quality DNA, presence of a smeared band indicates DNA
degredation.

Thank you for your suppport, mam..
I am sure i can count on you when i am in doubt...
I will reply with my progress in 12 hours...