**********
This page was written to fulfill a requirement for Genetics Lab at UMBC
**********
|
|
| |
|
|
| |
Cristina Casaje: Sec. 302, Writer, Editor, Liason
Venice Luceriaga: Sec. 203, Writer, Web Developer
Created December 14, 2004
|
|
|
|
Experiment: Streptomycin Mutations in
E. coli
Materials & Methods
The Mutation Frequency in E.coli : Streptomycin
The mutation frequency was determined by plating diluted 10^-6 E.coli on two TBAB plates and plating E.coli on one TBAB and Streptomycin plate. The following day the colonies were counted up. The calculations were done for the viable cell count, mutant concentration, and the mutation frequency. A more detailed description of what was done is found below in the diagrams below: “Preparation of TBAB plates” and “Preparation of TBAB and Streptomycin plate.” The TBAB and Streptomycin plate was saved for later use in the procedure.
After several weeks, a colony from the saved TBAB and Streptomycin plate was spread. A diagram on how to spread the plate with 3 toothpicks is shown below(“Streaking E.coli mutant from the saved TBAB and Streptomycin plate”). A streak that appeared on this particular plate was plated onto two new plates:TBAB plate, TBAB and Streptomycin plate. This procedure tested if the mutant E.coli was Streptomycin dependent or resistant.
Polymerase Chain Reaction
The rpsl gene was amplified by adding water, a left primer, a right primer, and E.coli to the PCR mix in a tube. The tube was placed in the thermocycler. The program of the thermocycler was as followed:
The PCR was cleaned up by mixing buffer PB and the PCR reaction. The mixture was placed in a Qiagen Column that was placed in a microfuge tube. This was spun in the microfuge and the liquid in the microfuge was emptied. Buffer PE was added and centrifuged. A new microfuge tube was used and water was added. This served as the purified PCR sample. 1 microliter of tracking dye was added to this sample and ran on an agarose gel.
The reaction was sequenced by mixing big dye premix, sequencing buffer, PCR product, primer, and water in a PCR tube. This tube was programmed as followed:
|
| | |
|
