BIOL 142 Lecture Notes - Lecture 11: Long Term Ecological Research Network, Gel Electrophoresis, Escherichia Coli
Tuesday, March 27, 2018
BIOL 142
•Construct cDNA library"
•Isolate plasmid containing fragment of interest from the cDNA library"
•Clone fragment/plasmid in E.coli"
•Isolate amplified fragment/plasmid"
•Express in E.coli or switch to eukaryotic host"
•Harvest Protein"
•E. Coli cannot do glycosylation because it doesn’t have an ER"
•Ligase - seals the connection between the insert and the plasmid"
•PCR - denaturation, annealing, extension"
•Taq polymerase - resists extreme temperatures, taken from archaea"
•Molecular searching for a clone - isolate molecules of interest, separating
biomolecules based on:"
•Size"
•Shape"
•Charge"
•Reactivity"
•Mass"
•Molecular searching techniques- southerns (DNA), northerns (RNA), westerns
(proteins)"
•General steps for molecular searching"
•Separate molecules by size"
•Transfer to solid support"
•Prepare molecules for probing"
•Probe for your molecule"
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