Experiment #: 8

DNA Isolation, Acid Hydrolysis, Determination of Concentration

via UV Spectronomy and Chemical Characterization

of Allium cepa ( white onion )

Oa, Bill Clinton N.

Pasion, Mark Lorenz S.

*Pureza, Beatriz Soneth M.

Ragudo, Gabriel Phillip P.

Department of Biological Sciences

College of Science, University of Santo Tomas

Espaa, Manila, Philippines

ABSTRACT

Keywords:

I INTRODUCTION

II METHODOLOGY

Materials:

Chopped, fresh white onion Ice-cold 95% ethanol

Homogenizing solution Tris-EDTA buffer or TE buffer

Standard Saline Citrate or SSC Cheesecloth

Commercial papain Blender

Procedure:

A minimum of two and maximum of three white onions were used in the experiment. The

few layers of the onions were removed while the remaining parts were minced and weigh up to

25 grams. 50 mL of homogenizing solution was added to the onions in a 125 mL Erlenmeyer

flask which was preheated to 60. It was then stirred and heated for 5 minutes. Three tablets of

powdered crude papain were added and then kept in 60 water for 10 minutes with constant

stirring. After which it was transferred to ice bath. It was then poured into the blender and

blended for 45 seconds. Using a cheesecloth it was filtered to 100 mL graduated cylinder. The

residue was discarded while the filtrate had its volume measured. It was then transferred to 350

mL beaker and put in ice bath. Then ice cold 95% ethanol which is twice the volume of the

filtrate was slowly added and allowed to settle for 5 minutes. The DNA that floats was spooled

using a glass rod and dropper and was transferred to a watch glass. The isolated DNA was air

dried, weighed and divided into two portions. The first division was 1.0 2.0 mg of DNA mixed

with SSC (3.3 mL SSC/mg) was placed in a large test tube while the rest of the DNA was placed

in a medium sized test tube covered with a cork and was stored inside the refrigerator.

The DNA with SSC was used to determine the concentration and purity. Quartz cuvette

was used to read its absorbance at 260nm an 280nm. The protein concentration and then nucleic

acid concentration was determined in the DNA solution. Also the absorbance ratio was computed

and from this the purity of the DNA solution was rationalized.

The DNA in a medium sized test tube was dispersed in 10 mL of 1 M of HCl and covered

with a marble. It was heated in boiling water bath for 60 mins and was cooled to room

temperature. 2.5 mL of distilled water that was neutralized with 1 M NaOH was added. Litmus
paper was used to check if it was neutral. Then it was filtered to a 10.0 mL graduated cylinder.

The residue was discarded while from the filtrate 4 mL was collected and transferred to a larger

test tube covered with cork for chemical characterization.

Four different chemical characterizations were done. First the test for Deoxyribose or

Dische reaction. This was done by adding 1.5 mL of diphenylamine reagent to 1.0 mL of the

DNA hydrolysate or to 0.5 mL deoxyribose standard solution. It was then heated for exactly 10

minutes in a boiling water bath, cooled immediately and observed the results. Second, the test for

H 2 SO 4
phosphate was done by adding 1.0 mL of concentrated to 1.0 mL of DNA hydrolyzate

HN O3
or to 1.0 mL of the phosphate standard solution. 0.5 mL of concentrated was added.

1.0 mL of distilled water also added and then it was heated for 5 minutes in a boiling water bath.

It was cooled then 1.0 mL of ammonium molybdate solution was added right after. It was mixed

well then diluted to 10 mL with distilled water. After 10 minutes the results was observe. Third

was the test for purines of Murexide test. This was done by placing a little amount of DNA

hydrolysate or the standard guanine or adenine solution in a small evaporating dish. Few drops of

concentrated nitric acid were added and evaporated to dryness very carefully in a water bath in a

fume hood. A yellow residue was observed and moisten with 10% KOH became red in color.

Upon further heating a purple red hue was observed. Few drops of distilled water were added

and then it was heated. Yellow solution was produced and later red residue upon evaporation.

Lastly, the test for pyridines or the Wheeler-Johnson test. 0.5 mL of DNA hydrolysate or small

amount of standard cytosine or uracil solution was treated with an excess of bromine water until

solution turns yellow. Then the excess bromine was removed by boiling the solution using a
water bath inside the fume hood until the solution turns light yellow to colorless. Barium

hydroxide in excess was added and tested with litmus paper. The purple color indicates cytosine

or uracil and is due to the purple barium salt of dialuric acid (2,4,6-oxy-5-hydroxypyrimidine).

And this test is negative for thymine.

III RESULTS AND DISCUSSION

IV CONCLUSION

V REFERENCES

Appling, D. R., Anthony-Cahill, S. J., & Mathews, C. K. (2016). Biochemistry: Concepts and

connections. England: Pearson Education Limited.

Campbell, M.K., & Farrell, S.O. (2013). Biochemistry. Canada: Cengage Learning.

Retrieved from: https://prezi.com/dvuyk47d7ihj/the-effects-of-temperature-and-ph/. February 19,

2017

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