* Antibiotic production * Recombinant DNA Restriction Enzymes * Bacterial enzymes: cut up foreign DNA * Specific: only but at recognition sequences * Palindromic: cut at the same base sequence on each strand‚ but in the opposite direction * The exposed bases provide “sticky ends” * H-bond to compliment bases of segments cut with same restriction enzyme Recombinant DNA 1. Isolate DNA from 2 sources * Bacterial plasmid w/2 genes * ampR(resistance to ampicillin)
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using a restriction enzyme digest; after which the cloned genes were inserted into the bacteria E. coli through the transformation process consisting of a restriction enzyme digest and ligation. Colonies of the bacteria were grown; plasmids from each colony were purified‚ and then
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DNA DIGESTION AND ELECTROPHORESIS In this experiment we will be doing a process called as DNA digestion or also known as restriction digest. A restriction digest is a procedure used in molecular biology to prepare DNA for analysis or other processing. It is sometimes termed DNA fragmentation‚ scientists Hartl and Jones describe it this way: This enzymatic technique can be used for cleaving DNA molecules at specific sites‚ ensuring that all DNA fragments that contain a particular sequence have the
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the concentration of DNA in the gel (Thermofisher Scientific‚ 2011). In this experiment‚ SYBRSafe is used for the detection of mutant OFP gene and the corresponding Plasmid DNA‚ pRSET vector after restriction digestion by KpnI and HindIII. Signal strength of SYBRSafe can be one of the parameters to measure the efficiency of restriction digestion. Q2. What is the purpose of the addition of ampicillin to the LB-agar plates?
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host. 2. A vector can be a plasmid‚ cosmid‚bacterophage‚retroviruses‚ animal and plant viruses or artificial chromosomes like YAC‚ BAC‚or HAC.(Yeast artificial chromosome‚ bacterial........) 3. The rec. DNA produced can be amplified or cloned in a suitable vector like bacteria for plamids‚ cosmids or bacterophages‚ plant and animal cells for viruses . Involves five steps: 1. Enzyme restriction digest of DNA sample. 2. Enzyme restriction digest of DNA plasmid vector (same Res.Enzyme). 3.
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in this particular lab was to isolate chromosomal DNA from mutants grown and observed in lab 5 and then digest the DNA using a restriction enzyme. The fragments left from digestion will be ligated and then transformed into a strain of E. Coli DH5αλpir containing the pir gene pi product for replication. This gene will allow only the plasmid containing the Tn to replicate. Afterward‚ the plasmid was isolated and sequenced into adjacent chromosomal DNA to determine which gene was interrupted by the
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Reactions of the DNA translocase FtsK and XerC & XerD recombinases from Pseudomonas aeruginosa Luhai Xu Student number 3107041 Supervisor Ian Grainge Table of Contents Abstract 3 LIST OF ABBREVIATIONS 4 Chapter1: Introduction 5 Chapter 2 Materials and Methods 26 Chapter 3: Results 41 Chapter 4: Discussion 63 References 74 Appendix 80 Abstract DNA replication is an important biological process and occurs in all living organisms
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Molecular Biology 344 fall 2012 A Johnson Due 9/11/12 as hard copy at the beginning of lecture Problem Set 1 1. Look carefully at the structures of the two molecules shown below. a. What would you expect to happen if you added ddCTP (shown in a) to a DNA synthesis reaction in vitro in large excess over the concentration of deoxycytidine triphosphate (dCTP)? How will this affect the pattern of bands on the sequencing gel? The ddCTP lacks the OH‚ which is required for elongation
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clone the lux operon by making Recombinant DNA and transform into another organism‚ E. Coli. Chromosomal DNA of vibrio fischeri was first extracted and digested with restriction enzyme Sal I‚ then ligated with the vectors and transformed into the E. Coli cells. A few white colonies indicating the E. Coli cells took up the hybrid plasmids were observed on the plate but no glowing colonies were detected. The lux operon was not successfully cloned in this experiment. Introduction Vibrio Fischeri possesses
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other to extract the DNA and RNA. After extracting the DNA‚ we it is important to use PCR amplification in order to amplify the DNA template to produce a specific DNA fragment. Another important step in DNA cloning is plasmid isolation. Plasmid isolation allows us to extract a plasmid from a bacterial cell (E.coli). In our experiments‚ we had to amplify either the 18S rRNA or the actin gene found in D. Melanogaster. Actin is a major contractile protein found in all eukaryotic cells‚ accounting
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