Pcr- Gel Electrophoresis

DNA EXTRACTION AND QUANTIFICATION

POLYMERASE CHAIN REACTION

AGAROSE GEL ELECTROPHORESIS TO VERIFY PRODUCTS

Introduction

Microorganism identification is used in a wide variety of applications including microbial forensics, criminal investigations and environmental studies. There are several methods for identification, such as molecular probes, restriction fragment length polymorphism, or Polymerase Chain Reaction (PCR). In this lab, we use PCR primers to target specific genes. In order to identify a type of microbe, there are many steps to go through including extracting genomic DNA (gDNA) from cultures, adjusting the concentration of DNA, doing a PCR reaction, and verifying amplification by gel electrophoresis.

The aim of DNA extraction is to extract intact DNA from the cells. Boiling cells in TE buffer is the simplest method to extract DNA. After extracting DNA, the DNA yield is measured by a spectrophotometer, and the concentration of gDNA is calculated for the PCR reaction. A thermocycler is used for PCR reactions. The three step PCR cycles contain denaturation, annealing, and extension. Agarose gel electrophoresis is the last step to verify PCR products. After separating DNA molecules by gel electrophoresis, ethidium bromide will stain both DNA and RNA to visualize the band patterns.

Materials and Methods

Egg, milk, spinach, Salmonella typhimurium are prepared to carry out the experiments. Salmonella typhimurium is positive control, and water is negative control. Diluting the DNA is 100 uL DNA and 900 uL H2O. Two ladders that are used are #1333 (1kb) and #0613 (50bp). Ladders and samples are added into ten wells (2 wells for each DNA sample). The order of samples is Spinach, Egg, Milk, Salmonella typhimurium, and Water.

In the exercise 6, 7 and 8, the procedures were followed in Molecular Techniques in Microbiology Labs by Boone (Pg. 51-52, exercise 6; pg. 57-58, exercise 7; pg.63-66, exercise 8)

Results

Exercise 6

Concentration of DNA

Dilution =...