Biology 475

Biology 475

Molecular Biology

Lab Two – Library Screening

With Restriction Enzymes

Rogan Rattray

Introduction

In this lab, we obtained plasmids from clonal E. coli host colonies that may or may not have the hot-spring bacteria PCR product successfully inserted into the plasmid. To digest those isolated plasmids and run through agarose gel electrophoresis to test for PCR product presence or absence.

Cloning Results

Methods: These clone samples are the result of special E. coli that have been “shocked” into taking up plasmids that hopefully have the PCR products inserted that were made during Lab 1. The bacteria were then grown on selective media with ampicillin to eliminate any cells that did not take up the plasmid (upper right). The selected colonies were then grown in liquid media as shown (right)

	Miniprep Methods
Methods: During the Miniprep process, the samples were spun in the microfuge several times in order to separate the plasmids from the other cellular components found in solution

	Restriction Methods and Results
Digestion Methods: After the cocktail containing the Eco R1 restriction enzyme had been added to the purified plasmid samples, they were incubated at 37 degrees C for about an hour.
Electrophoresis Methods: While the plasmid samples were being digested by the restriction enzyme, 1% agarose gel plates were prepared by dissolving 0.5g agarose powder 50mL of water for each plate. Since four plates were needed, 2g of agarose was dissolved in 200mL of water.
Results: After the cut plasmid samples were ran through the agarose gel, they were visualized by soaking the gel in ethidium-bromide and then exposing it to UV light. This gel (right) clearly shows lanes 1 through 11 (from left to right). Lane 1 has the weight standard sample in it. Lanes 2, 3, 6, 7, 9 and 10 correspond to samples 41, 42, 45, 46, 48 and 49. These lanes have two visible bands indicating that samples had a successful insert of 16S cDNA into the plasmid. The bands closer to the top of the picture are the plasmid while the lighter band near the bottom are from the 16S insert

Discussion

The procedures for plasmid isolation were not as rigorous as those for PCR, and they seemed more forgiving of errors. Of the ten samples that I was given to process and analyze, the gel showed six of them to have positive insert of PCR product. The gel cannot show whether all of these inserts were from Red mat bacteria or some other source. More sensitive tests will need to be done to determine that distinction.