Blue White Screening Report

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a. Blue-white screening: The mechanism of this method is based on the genetic engineering of the lac operon in the Escherichia coli strain that acts as host with a subunit shared by the cloning vector. The vector has the α subunit of lacz protein with the MCS (multiple cloning sites) and the host chromosome shares the rest of the subunit forming a β-galactosidase enzyme. Then the foreign DNA is inserted in the lacΖα gene, as the MCS can accommodate any restriction enzyme. So, this disrupts the production and functionality of the β-galactosidase. Xgal is the chemical required for the screening, is a modified, colorless gal sugar acts as an indicator when metabolized by the β-galactosidase. The colorless X-gal is hydrolyzed by β-galactosidase…show more content…
Colony PCR: Colony PCR is very commonly used high-throughput method to determine if the insert DNA is in plasmid constructs. Transformants can be created either by lysing it in hot water (heating step) or add it to PCR reaction to lyse it in initial heating step. Initial heating step release the plasmid DNA from the cell, to act as template for the amplification reaction. Primers are designed to exclusively target the insert DNA where that confirms if the construct contains the DNA fragment of interest. Some use primers targeting vector DNA insert that helps to evaluate if the insert has correct molecular size. Insert specific primers explains both the specificity and size of the insert DNA, vector specific primers allows screening of multiple constructs at a time. Insert orientation can also be determined by colony PCR. PCR amplification of the plasmid with both an insert and vector specific primers are designed to produce an amplicon of a specific size only if the insert is in the correct orientation. In all experimental designs, presence or absence of a PCR amplicon and size of the product are determined by electrophoresis alongside a DNA size marker on an agarose gel. However, sequencing is the best way chosen in present world to confirm the DNA insert in the

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