Recombinant Plasmids and Restriction Enzymes Introduction In this ex yeast two-h
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Recombinant Plasmids and Restriction Enzymes Introduction In this ex yeast two-hybrid system. Th transcriptional factor binding domain and the murine p53 protein. The pl inserted into the plasmid at the multiple cloning site (MCS) using BamH I and EcoR I insert is 974 bp long. Figure one shows the plasmid, pVA3, and indicates the size and position of the insert you will isolate plasmid DNA from pVA3, the yeast plasmid that we will use in the is plasmid contains the gene that includes the insert for the fusion protein, the Gal 4 asmid is 6.4 kb in size. The p53 gene is restriction enzymes. The Figure 1: Diagram of yeast plasmid, pVA3 Mind (as 72) EooR I BamH I PAD Murine p53 insert Pst I Nae I -Hind aa 390) pVA3 4 kb Aat II TRPI bp 1345 Amp Xba I Col E1 We will use a rapid method of plasmid DNA isolation that does not require phenol or chloroform for py- rification and is the method used in many research laboratories today. It is the Qiagen QlAprep plasmid miniprep method. The following procedure has been slightly modified and taken from the QIAprep Miniprep Handbook, QIAGEN, l 1 /2005. This protocol is designed for the purification of up to 20 g high-copy plasmid DNA from 1 to 5 ml overnight E.coli cultures. The method is a modification of the alkaline method of purification. In the alkaline tured by SDS and NaOH. Neutralization of the solution results in a fast reannealing of covalently closed plasmid DNA due to the interconnection of both single-stranded DNA circles. Much more complex bacterial chromosomal DNA cannot reanneal in this short time and forms a large, insoluble DNA network, largely due to interstrand reassociation at multiple sites along the long linear molecules. In the next step of the procedure, lowering the temperature results in precipitation of protein-SDS complexes. Subsequently both complexes, DNA and protein, are removed by centrifugation leaving plasmid molecules in the supernatant. The plasmid DNA can then be bound to a mini spin column and recovered by elution with water or dilute buffer After you obtain your plasmid DNA, you will digest it with restriction enzymes. The object will be to re- lease the 974 bp P53 gene insert by doing a double restriction enzyme digest with both EcoR I and BamH I.Since the insert was ligated into the EcoR I and BamH I sites, digestion with the same enzymes should release the insert from the plasmid. The result will be a linear plasmid (5426 bp) and the insert (974 bp). You will also digest the lysis method, cells plasmid DNA with EcoR I and BamH I alone. Each enzyme by itself should introduce a single, double-stranded cut in the plasmid and cause it to change from the supercoiled circular form to the linear form but will not release the insert. Digestion by both enzymes is necessary to release the insert. We will estimate the size of the linear plasmid with insert and the sizes of the plasmid and insert separated using agarose gel electrophoresis and DNA "The material in the spin column is proprietary but is robably a glass ther or silicon matcrial. 35 Recombinant Plasmids and Restriction EnzymesExplanation / Answer
The silica resin spin colums are modified through attachment of anionic group. Due to the high surface density of anion groups, the resins become highly selective for DNA. Alongwith this, the high salt and pH conditions are conducive to selective binding of plasmid DNA.
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