User:Klare Lazor/Notebook/Chem-496-001/2011/09/20

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Objective

mutation of GFP to contain a cysteine residue just after the enterokinase clevage site through PCR.

Description

Quick Change Manual was used in order to determine the forward and reverse PC primers. The PCR reaction was performed by using previously prepared primers. 43uL of ddH2 was added. 1uL on .5u/uL of template DNA, 1uL of a 10mM dNTP solution, and 1.25uL of a 100ng/uL solution of forward and reverse primers was pipeted into the solution. 1uL of enzymes was added to the solution, along with 25uL of wax to prevent evaporation, before placed in the thermocylcer.

Data

Primers: FORWARD: 5' GAC GAT GAC GAT AAG GAT CGA TGG GGA TCC GAA 3' REVERSE 5' TTC GGA TCC CCA CCA TCG ATC CTT ATC GTC ATC GTC 3' [edit]

Notes

PCR Background

  • Polymerase chain reation is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a DNA sequence. The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reation for DNA melting and enzymatic repilcation of DNA. As a PCR progresses, the DNA generated is itself used as a template, setting in motion a chain reaction of replication. All PCR's apply and enzyme for heat stability. The alternation between heating and cooling are necessary to separate the two strands in a DNA double helix (occurs at high temperature), and to use each strand as the template for DNA synthesis at lower temperatures.

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