Genetics Lab Report 2 (editied from 1).docx - Cynthia...

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Cynthia Deokarran and Firdous Ali 9/13/18-9/20/18 Transformation, Cloning and Analysis of pGLO Plasmid Abstract In this experiment, we added transformation mixtures with different types of plasmid DNA to E. Coli to observe the cloning and transformation properties. This experiment was conducted by incubating pGLO DNA with competent E. Coli , heat shocking the DNA mixtures and adding a growth medium. The cells were plated, incubated and checked for colonies. Our results showed that plate 7, containing uncut pGLO, LB, Amp and Ara was the only media plate to yield any fluorescent colonies. We concluded that Arabinose assisted heavily in allowing green fluorescent protein (GFP) to enter the cells to produce fluorescent colonies. Introduction DNA cloning is a process used to create new DNA by placing it in a host that will allow it to replicate itself. DNA is put into a vector which is then relocated to a host so that will allow it to replicate using the hosts’ cellular machinery. Once the DNA has replicated, it is purified so to allow for analysis with restriction enzymes to verify that it is the plasmid of interest. In this lab, our goal was to replicate pGLO with arabinose and ampicillin through transformation so that we could figure out which plates would yield colonies and of the colonies yielded, which of them contained DNA that could replicate with green fluorescent protein and which could not. To clone the different types of DNA used in this experiment, we mixed them with plasmid and put them on to E. Coli media plates to help them grow in colonies. Cloning the DNA allowed us to determine whether if uncut DNA along with the supporting operon Arabinose makes the colonies fluoresce. Methods and Materials The procedure used can be found in the 3100-212 Genetics Lab Fall 2018 Handout titled “Transformation, Cloning and Analysis of pGLO Plasmid.” The following changes were made: On day 1
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(9/13/18), 100 ng/uL TE (1/5 dilution) was provided to us lab by our TA. For microcentrifuge tube B in part A, only 0.05 ng/uL of pGEM-EZ was used instead of 0.1 ng/uL. PGEM 3Z was provided along with
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