FINISHING FIRST PCR EXPERIMENT AND INTRODUCTION TO MAKE GEL.

January 26^th 2012.

Today Mr. Adams had to leave early so I worked with Ms., Urban in the lab finishing to set up my PCR mix for my very first experiment. We used a 1ml tube to make the master mix before distributing it in smaller tubes. There were 5 small tubes plus 1 as a control group; we mixed dNTPs, Primers, Buffer, Water, MgCl₂ and the DNA in a 1.5 ml tube (Review my previous blog for details).

After mixing everything in and distributing it the small tubes (about 1.5microliters), Ms. Urban put the master mix tube on a machine that mixed all the ingredients well. Then she placed the tubes in a Thermo cycler where the mixtures will be heated to separate the DNA then cooled so complementary strands can attach to the original DNA; this process continues until there are no new strands to “copy”. The entire process takes about 2 hours, and she used a program called, Q1A42-57, a special program for the type of primers we used.

While the mixture was in the Thermo cyclers Ms. Urban gave me an introduction on how to make a gel where the DNA will be placed to see if the experiment worked. The gel is made from Agar powder. To make it, mix the Agar powder with Buffer then heat the mixture in the microwave to dissolve the agar. After wards, add “the red dye” that will show you if the DNA has been made and set the agar for about 30 minutes to cool into actual gel. While the Agar is cooling add “combs” that will make holes for you to put the mixture in. After the gel has cooled, ass the PCR mixture, with “the blue dye” to the spaces made by the combs.

All the while Ms. Urban was instructing me on the gel, another researcher was making his gel, so I had the advantage of watching someone make the gel. After adding the PCR, the gel is placed in an Electromagnetic machine that makes the DNA flow based on the charges present in the gel and the DNA. This process takes about 5 minutes. Afterwards the gel is done a picture is taken under UV light with a special camera to see how the DNA places itself on the gel.