Polymerase Chain Reaction Lab Report

Materials & Methods To determine the presence of tissue plasminogen activator (TPA) regions on chromosome 8, we prepared a polymerase chain reaction (PCR) to amplify our DNA samples and used gel electrophoresis to visualize the results. Samples of cheek epithelial cells collected by rinsing our mouths with 10 mL of a 0.9% NaCl solution for 30 seconds were used as the template DNA for the reaction. Using a 100-1000 μL pipettor, two increments of 750 μL of the expelled salt and cheek mixture were transferred into a labelled 1.5 mL microfuge tube. Tubes were collected and centrifuged at 13 000 rpm for 10 minutes. Following centrifugation, the supernatant in the tube was discharged into the sink and the tube was placed in an ice bath.

Using a 100-1000 μL pipettor, and swirling a bottle of 10% Chelex continuously, 500 μL of the cloudy Chelex mixture was added to the remaining cell pellet in the tube and resuspended to confirm that no clump of cells remained in the solution. After being placed into a 100°C heat block for 10 minutes to release the DNA trapped in the cell pellet, the tubes were immediately placed in the ice bath for one minute then spun at 13 000 rpm for three minutes then returned to the ice bath. Using a 1-10 μL micropipettor, 10 μL of supernatant containing the DNA was removed and placed into a labeled PCR tube. A 10 μL-100 μL micropippetor was set at 40 μL, and the appropriate amount of Master Mix, having been kept on ice for the entirety of the procedure, was removed and added to the tube. All completed samples were placed in an ice bucket, after which they were loaded into a PCR machine and ran following the

One flask of 1.5% agarose was removed from a 65°C water bath and poured into the tray once it was cool enough to hold. The 1x TBE buffer was used to fill the chamber in the gel box to adjust the buffer to point at which it sits 1 cm over the middle level. After having removed the comb, a 1-10 μL micropipettor was used to remove 10 μL of the 2-Log DNA Ladder provided by New England Biolabs (NEB) mixed with a loading dye, giving it a distinct blue color. The DNA ladder contained DNA fragments of known sizes, which allows us to measure how large our DNA fragments in comparison to the provided DNA ladder. A 1-10 μL micropipettor was used to extract 5 μL of the loading dye, which was then added to the PCR mixture and mixed gently. A 10-100 μL was used to remove 20 μL of the PCR mixture and loaded into one of the wells. Each group was also assigned a control, of which 20 μL was loaded into one of the wells. Four controls were used to ensure that the procedure was performed correctly and efficiently. No template DNA was added to demonstrate that no contamination had occurred during the procedure. No Taq polymerase was added to ensure that no spontaneous assembly of DNA had materialized. Taq polymerase was used an alternative to DNA polymerase as it can withstand higher temperatures and does need to be added as frequently to maintain PCR reactions. Without the