• Objective
  • Understand the concept of PCR (polymerase Chain Reaction) Protocol and perform the PCR with the extracted ciliate DNA, prepare a gel for electrophoresis
• Background
  • DNA from ciliates was extracted in the previous lab via a modified chalet extraction method
  • PCR is a process used in biology that amplifies certain genes from the DNA by denaturing, annealing, and elongating the gene (See Figure 1)

  Figure 1: PCR Protocol 

• Procedure
  • PCR Protocol
    • Create the experimental group by combining 12.5 µL AmpliTaq Gold 360 Master Mix (Polymerase, nucleotides, and buffers), 5 uL DNA (the concentration of DNA ~ 10-100ng), 1µL of the 20 µM primer mix (both primers are in this mix), and 6.5 uL water in a PCR tube
    • Vortex the tube for 5 seconds to ensure all of the reagents are at the bottom of the tube
    • Create the control group by mixing all of the same experimental group amounts of chemicals, except for including DNA. The amount of volume of DNA put in the experimental group should be replaced by an equal volume of water (6.5 uL)
  • Conduct the thermocycling procedure by conducting the following cycle steps (conducted by lan instructors)
    • Initial denaturation: 95° C for 2.5 min
    • 35 cycles:
      • Denaturation: 95° C for 30 s
      • Primer annealing: 56° C for 20 s
      • Primer elongation: 72° for 2 min and 30 s
    • Extension: 72° C for 5 min
  • Gel Preparation
    • 1.5 g of agarose was added to 100ml of 1xTAE in a 250ml flask, and then was heated in a microwave until dissolved completely
    • Mix the agarose solution until cooled
    • Ethidium bromide comb was inserted into the gel tray, and the cooled agarose gel was poured until the tray was approximately half full
• Data
• Further Discussion and Experiments
  • The PCR protocol was used prior to the gel electrophoresis to amplify the DNA strand, and compare it to the S18 strand to hopefully determine if we successfully extracted ciliate DNA from the soil sample
  • The next steps include running the Gel electrophoresis which includes inserting the DNA into a well and adding an electrical current that will distribute the DNA across the gel, which will then be compared to the DNA ladder of the S18 gene