*4.6.1 Genomic DNA extraction of isolated bacteria*

For genomic DNA extraction, the bacteria isolated, that has pure colony on a plate was washed with 2 ml of TE buffer. Then, 1 ml of the mixture was transferred into new Eppendorf tube and centrifuged at 10000 rpm for 2 minutes in order to obtain pellet. Next, the pellet will be resuspended with 500 μl TE buffer, vortex until pellet dissolved and divided into two tubes with 750 μl in each tube. Then, 65 μl of 10% sodium dodecyl sulphate (SDS) and 10 μl proteinase K were added before incubating for 1 hour in incubator and flipped every 10 minutes. Next, 825 μl of phenol/chloroform/isoamyl alcohol (25:24:1) was added and placed on shaker for 45 minutes. After that, the tubes were centrifuged for 15 minutes at 10000 rpm until double layer is formed. Then the upper layer of the mixture was extracted into new sterile 1.5 ml tubes. Next, 200 μl of isopropanol will be added before spinning for 15 minutes at 10000 rpm and the tubes were flipped a few times. Once the pellet of DNA was obtained, the supernatant was removed, and the pellet was air dried before adding 50 μl TE buffer to dissolve the pellet and stored at 20 °C for further analysis. Then, the extracted genomic DNA was also analyzed by using agarose gel electrophoresis to confirm presence of DNA.

#### *4.6.2 Polymerase chain reaction*

Polymerase Chain Reaction (PCR) was carried out by using universal primers which is forward primer 27F and reverse primer 1492R to amplify 16S rRNA gene. **Table 3** shows the sequence for PCR.

All PCR reagents were pipetted into 0.5 μl Eppendorf tube and mixed gently to make sure all mixtures were collected at the bottom of the tube. **Table 4** shows the reaction mixture used for polymerase chain reaction. Next, the tubes containing


**Table 3.** *Polymerase chain reaction universal primer [35].*


#### **Table 4.**

*Reaction mixture for polymerase chain reaction.*


**Table 5.** *PCR profiles.*

PCR mixture were placed in DNA thermal cycler machine and performed according to parameters shown in **Table 5**.
