ABSTRACT

The destination from studying genomic was to know how the structure DNA and

how to processing analyze DNA. The Genomic experiments consist of Blood

Separation, DNA Isolation, Quantitation of DNA concentration, DNA detection using

electrophoresis, Polymerase Chain Reaction, and DNA Sequencing.

The first experiment is Blood Separation which intended for separating blood

components that will be used later for DNA extraction and others process. It was

resulted in three and two layers that contain plasma and serum. Then, DNA Isolation

intended to isolate DNA from human blood (White Blood Cell) and understand principle

of DNA isolation technique. Next, Quantitation of DNA Concentration which be used to

determine of DNA concentration using NanoDrop, calculating the absorbance of DNA,

and determine the purity DNA that isolated. DNA is considered pure if the purity index is

1.80-2.00. Later, DNA detection using electrophoresis procedure that describes the

method of double helix DNA detection was done to make sure whether the procedure

of DNA Isolation was successfully done, using agarose gels to separated DNA in the

range of 200bp-50kb. Next, Polymerase Chain Reaction was done to amplify ERCC 2

gene (330 bp) sequence which furthermore will be used in DNA Sequencing. PCR

performs the chemistry of DNA duplication in vitro was technique for replication or

copying a portion of a DNA strand outside a living cell. Last, DNA Sequencing using

BLAST for determination of the precise sequence of nucleotides in a sample of DNA.

I. INTRODUCTION

II. MATERIALS AND METHODS

II.1 Blood Separation

Materials : Blood, Vacutainer, Syringe, Vials, and Centrifuge

Blood samples were drawn 8-9 ml from Kevin and Rio and stored in vials. Giving

label each vial properly. 1 ml of whole blood is transferred into a separated vial. Then,

centrifuge the vacutainer at 3000 g, 20oC, for 10 minutes. Transfer the plasma into a

new vial. The vials were stored at -80°C.

II.2 DNA Isolation

Materials : Ethanol absolute, Isopropanol (2-propanol), Proteinase K, Sodium

acetate, Sodium dodecyl sulfate (SDS 10%), Tris EDTA buffer with pH 8.0,

Chloroform / lsoamyl alcohol 24:1 which contained 24 ml of Chloroform and 1 ml

of Isoamylalcohol, and Proteinase K (10 mg / ml).

Firstly, blood samples typically were obtained as 0.5 ml of the whole blood stored in

EDTA vacutainer tubes, then frozen at -70°C. Add 0.8 ml 1X SSC buffer, mix, centrifuge

for 1 minutes at 12.000 rpm, after centrifugation remove 1 ml of the supernant and

discarded into disinfectant. Next, 1 ml of 1X SSC buffer was added and vortex,

centrifuge for 1 minutes again, remove all of the supernatant. Next, 375 μl of 2.0 M

NaOAc was added to each pellet and vortexed briefly. Also, 25 μl of 10% SDS and 5 μl

of proteinase K (10 mg/ml H2O) were added. Then, vortex and incubate for 1 hour at

55°C. After that, 120 μl of phenol / chloroform / isoamyl alcohol ( 25:24:1) was added

and vortex for 30 seconds. Centrifuge for 2 minutes. The aqueous layer was carefully

removed to a new 1.5 ml microcentrifuge tube. After 1 ml of cold 100% ethanol was

added, mixed, and incubated for 15 minutes at -20°C, centrifuge again for 2 minutes,

decant the supernatant, and drained. Then, 180 μl of 1x TE buffer was added, vortex,

and incubated at 55°C for 10 minutes. Afterwards, 20 μl 2 M Sodium acetate and 500 μl

of cold 100% ethanol was added, was mixed, centrifuged for 1 minute. Thereafter, the

supernatant was decanted and the pellet was rinsed with 1 ml of 70% ethanol,

centrifuge for 1 minute. The supernatant was decanted and the pellet was air dried for

10 minutes or until dry. The pellet was then resuspended by adding 200 μl of 1X TE

buffer, incubated at 55°C for 30 minutes, vortexed periodically to dissolve the genomic

DNA. Lastly, the sample was stored at -20°C.

II.3 Quantitation of DNA concentration

II.4 DNA electrophoresis

II.5 Polymerase Chain Reaction

II.6 DNA Sequencing