Lab Report Polymerase Chain Reaction

Polymerase Chain Reaction (PCR) Abstract Polymerase Chain Reaction (PCR) was performed with DNA template of pBR322. The PCR ran for approximately 25 cycles (90 minutes). The prepared sample, after the PCR, was loaded into a polyacrylamide gel electrophoresis. The results specify a migration distance of 6.3 cm and 316 base pairs. The error is 5.33% due to a possible inappropriate positioning of the gel. Introduction Polymerase Chain Reaction (PCR) is a technique used to amplify a single or multiple copies of a piece of DNA. A PCR have three components: a pair of primers, all four-deoxyribonucleoside triphosphate (dNTPs) and a heat stable DNA polymerase. A PCR primer is a short fragment of single stranded DNA that is complementary to DNA sequences of the target region (University of Toledo, 2004). The primers help provide a free 3-OH group to which the DNA polymerase can add dNTPs (University of Toledo, 2004). The dNTPs (dATP, dCTP, dGTP,dTTP) are nucleotide base pairs that are added to the DNA strand by DNA polymerase. The final important component of PCR is a heat stable DNA polymerase, Taq DNA polymerase. Taq DNA polymerase can synthesize DNA under heat intensive condition because Taq is isolated from the bacterium Thermus aquaticus that lives in hot environments (University of Toledo, 2004). The PCR cycles involve three steps: denaturation, annealing, and extension. The denaturing step involves heating the solution to 90 °C to denature and separate the two strand of the parent DNA molecule (University of Toledo, 2004). The denaturation is followed by annealing, which involves the cooling of the solution to 54 °C to allow the primer to hybridize the DNA strand (University of Toledo, 2004). The first primer hybridize, the 3’ end of the target strand, while the second primer hybridizes to the 3’ end of the complementary target strand (University of Toledo, 2004). The final PCR step is extension. Extension involves heating to 72 °C, the ideal temperature for heat stable polymerases (University of Toledo, 2004). The polymerase, Taq DNA, elongates both primers in the direction of the target sequence (University of Toledo, 2004). DNA synthesis takes place on both strands, however it extends beyond the target sequence (University of Toledo,2004). Procedure In a small centrifugal tube add the following: 5 μL 10x PCR buffer 3 μL 25mM MgCl2 2 μL Template DNA 1 μL dNTPs 2 μL forward primer 2 μL reverse primer 2 μL Taq DNA polymerase 33 μL H2O The solution was added to a total of 50 μL. The solution ran for 25 cycles (90 minutes) in a Polymerase Chain Reaction (PCR). A polyacrylamide gel was prepared during the 90 minutes of the PCR reaction. A 50 mL of 30% acrylamide/bis-acrylamide was prepared with a weight ration of 29:1. Afterward, 60 mL of 6% polyacrylamide gel was made with the following: 35.5 μL H2O 12 mL of 30% acrylamide/bis-acrylamide 12 μL 5x TBE 480 μL APS 20 μL of TEMED An important final step, 20 μL of TEMED, was added lastly. After the PCR cycles were finished, the loading sample was prepared with 10 μL of the reaction mixture, 3.3 μL of dye, and 6.7 μL TE buffer. The prepared sample was loaded into the polyacrylamide gel. Log(DNA Basepairs) Results Log(DNA size) vs Migration Distance 3 2.5 f(x) = − 0.117983532920077 x + 3.24311504671905 R²2 = 0.99771886989741 1.5 1 0.5 0 3 4 5 6 7 8 9 10 11 Migration Distance (cm) Migration Distance 4.1 4.6 5.4 6.3 8 10.6 Log(Base Pairs) 2.77815125 2.698970004 2.602059991 2.477121255 2.301029996 2 Lane 6 y = -.118x +3.2431 Our DNA has 316 base pairs compared to the Lane Marker of 300 base pairs. Migration Distance of Lane 6 is 6.3 y = -.118(6.3 cm) + 3.2431 y= 2.4997 102.4997= base pairs base pairs = 316 bp Percent Error: ⌈ 300−316 ⌉ x 100 % = 5.33% 300 Discussion Polymerase Chain Reaction (PCR) was used to amplify the DNA template of pBR322. The primers used were: 5’-GGCAGGTAGATGACGACCAT-3’ forward primer and 5’TTTGCGCATTCACAGTTCTC-3’ reverse. The PCR ran for a total of 25 cycles for 90 minutes. The amplified DNA was loaded in a polyacrylamide gel. Lane 6 travelled 6.3 cm and has approximately 316 base pairs compared to the lane marker of 300 base pairs. There is 5.33% error, which is relatively low. However, the error could have resulted from inappropriate positioning of the gel, which could result in slanted bands. Consequently, it would be difficult to measure the migration distance, thus miscalculating the number of base pairs.