Biology 475 Molecular Biology Lab Two – Miniboiling prep Shanna Briggs Copyright 2003

Introduction  Between weeks 1 and 2, our instructors individualized the mixture of DNA resulting form the PCR done in week 1. This DNA was cloned into E. coli using a plasmid. During week 2 we isolated the plasmids, screened the library using gel electrophoresis.

	Cloning Results
Clone Library: This is the plate of numbered clones. We definitively know that these clones had taken up plasmid as indicated by the fact that they grew in media with ampicillin (the plasmid has ampicillin resistance gene on it). In addition, they are white, indicating that the lacZ gene has been interrupted by the insert.

	Mini-Prep Methods
Methods: We added lysozyme to digest the cell wall and centrifuged for 10 minutes to collect cellular debris and genomic DNA into a “snot pellet” which we removed with a toothpick.
Methods: We then added sodium acetate and isopropanol to precipitate the plasmid DNA. To assist this process, they were cooled at -65 C for 10 minutes.

	Restriction Methods and Results
Digestion Methods: After adding the restriction enzyme EcoR1, the samples were incubated at 37 C for 45 minutes to facilitate the activity of the enzyme.
Electrophoresis Methods: A loading dye was then added to the DNA to help with visualization and to weigh down the DNA. The samples were loaded on a 1% agarose gel and run at 125 V for 45 minutes.
Results: The Gel shows two bands for clones numbers 81-88 and 90. Clone number 89 does not show a plasmid or insert. The lane labeled “S” is the standard markers.	S 81 82 83 84 85 86 87 88 89 90

Discussion

The overall goal of this lab was to screen the clone library using gel electrophoresis. The clones, generated by heat-shocking special E. coli, were numbered for reference if they proved to have taken up relevant DNA inserts. To identify such clones we used two procedures. The first was to cut the isolated plasmids with the restriction enzyme EcoR1, which will make two cuts in the plasmids used. If the insert is present in the plasmid this will result in two fragments and thus two distinct bands on the gel electrophoresis. If the plasmid did not contain an insert, the result will be a single fragment and one band on the gel. In the section that I processed, all of the clones except number 89 had two distinct bands on the gel, indicating that they had a plasmid with the insert.

The significance for the project of this work is that we have identified clones that have DNA sequences from the original hot springs populations. Therefore, two clones with definite insert bands will be preserved for later analysis including DNA sequencing. These sequences will be used to assay red bacteria diversity in the original mat population.