Bacterial Genetics Lab Report

Bacterial Genetics

Aim:

The purpose of the lab was to alter the plasmid DNA of bacterial cells and to observe any variations in the phenotype of the bacteria expressed in the plasmid after incorporating new genes. The lab consisted of three parts. In the first part, plasmid DNA was incorporated into bacterial cells. The second part consisted observing the new phenotypic traits on agarose plates, and isolating the plasmid DNA from the transformed bacterial cells to be used in PCR reactions. The final part was analyzing the PCR reactions on agarose DNA gel electrophoresis.

Materials and methods:

Part 1 - Bioluminescence Materials:

• sharpie

• 37 oC water bath

• Ice

• Sterile transfer pipette

• Foam tube rack

• Transformation solution

Determine the concentration of DNA using a nanodrop apparatus. Dilute plasmid DNA to 20 µg/mL by following directions from the instructor. 2) Materials: Assembly of PCR reaction

• Edvotek PCR pellet

• Small PCR tube

• Micropipettors and tips

• Primers

• Plasmid DNA from Week 6

• Microcentrifuge

• Thermal cycler

• 10X gel loading solution

Procedure: Activity 2

1. Put on appropriate lab safety attire and gloves

2. Add Edvotek PCR pellet to 0.2 mL PCR tube

3. Add 20 µL of the primer pair.

4. Add 5 µL of plasmid DNA

5. Dissolve pellet by mixing. Put the tube in a microcentrifuge to spin and gather the remains at the bottom of tube.

6. Write initials on the tubes with sharpie.

7. Put the tubes on a thermal cycler and start it.

Part 3- Agarose gel electrophoresis

Materials:

• Agarose gel electrophoresis apparatus

• 1% agarose gel in 1X TAE buffer & SYBR® Green

• Microipettors and tips

• UV transilluminator

• PCR samples with added tracking dye

Procedure:

1. Put on appropriate lab safety attire and gloves

2. put 20µL of PCR reaction into a section in the agarose gel with a micropipettor.

3. Load DNA ladder in one of the agarose gel wells.

4. put agarose gel at 100 volts and run for an hour.

5. Observe agarose gel on UV transilluminator to examine results of the polymerase chain

The first step was making the cell competent so it can take up DNA. Then plasmid DNA was introduced to the cell and the process of transformation was underway. We then viewed any phenotypic traits. We look for growth and bioluminescence in the cells. Both positive plates with amp displayed growth and one of them was bioluminescent because of the ara presence. The two negative plates had no bioluminescence and the plate with amp had no growth. The final part of the objective was also met because we examined the PCR reactions on the agarose gel electrophoresis. The whole experiment can be applied to professional life in the future because the lab gave me a glimpse of what it means to be a scientist. In the future, I plan on becoming a doctor and members of the healthcare environment are thought to be scientists. This lab was just a small piece of what is going to prepare to become what I want in the near