Laboratory 21 DNA Part II (Electrophoresis and PCR)

Introduction

The DNA extraction protocol that you used in part 1 of this lab should have resulted in samples containing the full genomic DNA of Ps. aeruginosa. In order to visualize the DNA, and to measure its size, we will be carrying out a simple gel electrophoresis. Electrophoresis separates DNA according to its size by drawing it through an agarose gel using an electric field. DNA is negatively charged so it is attracted to the positive electrode in the chamber. As it moves through the agarose gel, larger pieces of DNA will be slowed down more than smaller pieces. The absolute size of the DNA fragments is estimated by comparing them to known standards. After we have confirmed that DNA is present we will perform a PCR amplification to isolate and detect the 16S rRNA gene from the genomic DNA. The 16S rRNA gene codes for a part of the ribosome and is present in all bacteria and archaea. Differences in the DNA sequence of this gene can be used to distinguish between different phylogenetic groups. PCR works by using short pieces of DNA (primers) that are homologous to parts of the sequence of interest to locate a specific gene. Once the primers bind to the target sequences, the stretch of DNA between them is copied. This process is repeated through many cycles, and results in an exponential increase in the numbers of copies of the targeted gene. The PCR product will also be visualized by electrophoresis and staining to confirm success of the reaction.

Materials

Equipment - thermal cycler Supplies - gel electrophoresis power source - 1% agarose gel with ethidium - electrophoresis rig bromide - micropipettes - TAE buffer - UV light box - sterile water - PCR kit - Bacteria specific primers - loading dye - DNA size ladder Procedures

Electrophoresis of Genomic DNA

1. Mix each sample with loading dye (1l dye with 5l sample) on a sheet of parafilm. 2. Place the 1% agarose gel into the electrophoresis box and cover with cold TAE buffer. 3. Load all 6l of each sample (including a size standard) into consecutive wells of the gel. 4. Place the cover on top of the electrophoresis box (check that the wires are attached to the correct electrodes). 5. Set the power supply to run for 30 min. at 125V. 6. After the gel has run, remove it and transfer to the UV light box to visualize the DNA bands.

1. Carefully label 2 PCR tubes for each DNA sample, 1 for the DNA and 1 as a negative control. 2. Transfer 19 l of the “dNTP mix” into each tube. 3. Transfer 10 l of the “27f primer” into each tube. 4. Transfer 10 l of the “1390r primer” into each tube. 5. Transfer 10 l of the “Taq polymerase” into each tube. 6. Add 1l of the DNA sample to the appropriate PCR tube and 1l of sterile water to the negative control. 7. Load the samples into the thermal-cycler and start the program.

Electrophoresis of PCR Product

1. Mix each sample with loading dye (1l dye with 5l sample) on a sheet of parafilm. 2. Place the 1% agarose gel into the electrophoresis box and cover with cold TAE buffer. 3. Load all 6l of each sample (including a size standard) into consecutive wells of the gel. 4. Place the cover on top of the electrophoresis box (check that the wires are attached to the correct electrodes). 5. Set the power supply to run for 30 min. at 100V. 6. After the gel has run, remove it and transfer to the UV light box to visualize the DNA bands. Standard protocol (for each 50  l reaction)

40.75 l PCR grade water 1.0 l dNTP mix (10mM each) 1.0 l 5’ primer (~5 pmol/l) 1.0 l 3’ primer (~5 pmol/l)

5.0 l 10X NovaTaq buffer with MgCl2 0.25 l (1.25 U) NovaTaq DNA polymerase 1.0 l DNA template (~10 ng)

Temperature Cycles

Melting 95C 5 min. Denaturing 94C 30 sec. Annealing 55C 60 sec. Extension 72C 90 sec. Final Extension 72C 10 min.

TAE Buffer

4.84 g Tris Base 1.14 ml glacial acetic acid 2 ml 0.5 M EDTA

Agarose gel

1% agarose dissolved in 1X TAE

PCR Primers

Bacterial (16S) ~1363 Bases 27f 5′ AGA GTT TGA TCC TGG CTC AG 3′ 1390r 5′ GTT TGA CGG GCG GTG TGT RCA A 3′ Results:

Was your PCR successful? Did your negative control amplify?

Questions:

What is the purpose of the dNTPs in the PCR reaction?

What is the purpose of the primers in the PCR reaction?

Would the PCR product look different if we started with mixed DNA instead of the pure culture?

List two things that may have interfered with the PCR reaction.