*4.6.3 Agarose gel electrophoresis (AGE), DNA purification and sequencing*

Agarose gel 1%was prepared for gel electrophoresis where o.75 g of agarose powder was added to 50 ml of distilled water in a conical flask which is then heated for about 40 seconds in microwave until agarose powder is fully dissolved. Next, 1 ml of 50X TAE buffer was added to the mixture and the mixture was poured into agarose gel electrophoresis tray with comb to let it solidify. After about 30 minutes when the gel is solidified, the PCR products and was loaded into respective wells and the gel was run at 100 V,400 mA for 30 minutes. After 30 minutes, the gel was stained with ethidium bromide for 10 minutes and then rinsed with distilled water for 5 minutes. Then, the gel is viewed under the UV light to observe the DNA band.

Once bands were observed, the PCR products were purified using DNA purification kit. Firstly, 20 μl of PCR mixture was mixed with 35 μl of binding buffer and the mixture was then transferred into a high pure filter tube that had been inserted into a collection tube. Next, the tube was centrifuged for 15 seconds at 5000 rpm and then the flowthrough in the collection tube was discarded. Then, 800 μl of washing buffer was added into the high pure filter tube which was then centrifuged at 5000 rpm for 15 seconds. The flowthrough in the collection tube was then discarded. Washing using buffer was done twice to enhance the result. Next the collection tube was replaced and the high pure filter tube was inserted into another sterile collection tube. Then, 30 μl of elution buffer was inserted into high pure filter tube and centrifuged for 15 seconds at 5000 rpm. The flowthrough, which was the purified PCR product in the collection buffer was collected and transferred into new 1 mL tube. Then, the purified DNA was sent to First BASE Laboratories Sdn. Bhd. Company for sequencing and the sequence data obtained were analyzed using MEGA software and BLAST from NCBI [36].
