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Polymerase Chain Reaction

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Biochemistry 2280A
Eric Ball

November 25 NotesHow do we purify the PCR product Did PCR workExpressing YFP in Ecolipurifying itHow do you identify and resolve DNA molecules DNA molecules are most easily separated based on difference in length number of base pairsGel Electrophoresis separated DNA DNA is sieved through a matrix of agarose or acryladmide gel separates the DNA based on length in gel there are very small pores the DNA goes through these poresDNA from each PCR reaction is loaded into a well in the agarose gel The gel is in a chamber in which an electric field can be appliedWhen placed in an electric field at pH7 DNA will migrate towards which pole PositiveWhen an electric current is passed through the chamber DNA fragments are pulled through the agarose matrix and move towards the positivepole Smaller DNA fragments move faster than large DNA fragmentsExcise the right oneDetection of the DNA in the gel1 Stain with ethidium bromide Eth Br fluoresces red under UV light when bound to DNA easily detectbands this waywhen you have little amounts of DNA this method wont workonly good with large amounts 2 Autoradiographyradioactively label the 5 ends of the DNA fragments using polynucleotide kinase and 32P ATP Expose yor gel to an Xray film figre 103What do we use as the template for the PCRIf our goal is to express YFhumanG would we use genomic DNA as the template No We use cDNA to PCR YFG Introns are a problem in expressing eukaryotic genes in Ecoli Ecoli can not splice them out cDNA cDNA is a replicate DNA copy of mRNA ccomplimentary
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