Random Amplified Polymorphic DNA

I learned  about another interesting part of my project during this week. I started to work on DNA which I haven’t done any experiment related to DNA since I started doing my internship in Biosciences Department. I extracted DNA of Agrobacteria. The first step is Random Amplified Polymorphic DNA (RAPD) markers. RAPD technique is based on the polymerase chain reaction (PCR) and this technique is of the most common molecular techniques to develop DNA markers. RAPD (Random Amplified Polymorphic) markers are amplification products of anonymous DNA sequences using single, short and arbitrary oligonucleotide primers. The Agrobacteria that I used was cultured onto TSB (Tryptic Soy Broth) broth and incubated at 37°C for 12 to18 hours. I poured 2ml of my bacteria into a centrifuge tube and centrifuged it in 4000 g for 2minuts. Then, I washed in 0.85% NaCl solution which I made it before and centrifuged my solution in 4000 g for 2minuts again. After that, I added Add 1 ml of TE buffer (EDTA 1.0 mM; Tris-HCl 10 mM, pH=8.0) to the bacteria and it was boiled in water bath for exactly for 15 minutes. I stared to add 10 microL of Master Mix to the 0.5mL Safe-Lock microcentrifuge tubes then 5 microL of Water RNA/DNA was added to the tube. The third substance that was added was 4 microL of primer (primers number one, two, six, and seven were used) and the last one was 1 microL of DNA polymerase.

A primer is a strand of nucleic acid that serves as a starting point for DNA synthesis. They are required for DNA replication because the enzymes that catalyze this process, DNA polymerases, can only add new nucleotides to an existing strand of DNA. The polymerase starts replication at the 3'-end of the primer, and copies the opposite strand (Wikipedia.) In the polymerase chain reaction (PCR) the primers has an important role. Primers allow the polymerase to start, because it can't just start by itself on a single stranded piece of DNA.

At the end, I placed my microcentrifuge tubes in the thermal cycler (also known as a Thermocycler, PCR Machine or DNA Amplifier). It took around three hours to whole cycle was completed. 

Standard amplification conditions were 94°C for 4 min followed by 44 cycles of 94°C for 1 min, 36°C for 1 min, 72°C for 1 min and the reaction was completed by a final extension step of 72°C for 10 min. Then, I placed my microcentrifuge tubes to the incubator to use them for next part of DNA extraction.