Genetic Defect Lab

 Part 1: Introduction

In this lab, we will be using a Polymerase Chain Reaction to see if a certain pattern of DNA is present in our cells. PCR has a wide range of uses, such as gene mapping, cloning, DNA sequencing, and the one we will be using, gene detection. The way this is done is by taking a DNA sample, and amplifying the gene of interest through PCR.

 Part 2: Experiment 

     On the first day, we will chew our cheeks and rinse our mouths with a saline solution and put it in a cup. Then we'll transfer 1ml of it into a micro test tube with 200 microliters of InstaGene matrix. Then we'll spin it in a centrifuge for 2 minutes. Then we'll pour out the saline solution while keeping the DNA pellet at the bottom of the tube, and flick the tube to resuspend the pellet. Then we'll put all of the resuspended cells into the screwcap tube with InstaGene using a 20 microliter micropipet, and throw out the flip-top tube that formerly contained the pellet of DNA. Then we'll shake or vortex the screwcap tube. When we're done, we'll put our tubes in a waterbath at 56 C for 10 minutes, shaking them after 5 minutes, then putting them back. Then we'll put the tubes in a 100 C waterbath for 5 minutes. Then we'll vortex the tube for a couple minutes and put it in the fridge until day 2.

     On the second day, we'll get our screwcap tube and spin it in a centrifuge. Then with a 20 microliter pipette, we'll extract part of the "supernatant," which is the liquid above the DNA pellet.Then we'll put 20 microliters of the yellow master mix into the PCR tube and mix it. Then we'll put the PCR tube in the thermal cycler for 40 cycles. We'll make sure to label our tubes and record their location in the cycler.

     On the third day, we will get our PCR tube and centrifuge it for 3 seconds. Then we'll add 10 microliters of PV92 XC loading dye and mix it gently. Then we'll cover the gel on the electrophoresis apparatus with 1x TAE buffer; about 275 mL of it. Then we'll load the lanes with the samples indicated in the packet. Then we will run the electrophoresis process at 200v for 3 minutes or 12 minutes, depending on period. Then we'll put the gel into the plastic staining tray and await further instructions.

  Part 3: Discussion