BMB Lab Exam II
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12 Terms
How is recombinant DNA technology used to produce proteins of interest?
To produce large quantities of proteins like insulin
What is recombinant DNA and how is the protein expressed?
A plasmid with essential genes and GOI; bacteria will read and express protein
What is a plasmid?
Small, unnecessary circular DNA found in bacteria, may give advantages
What are the parts of the plasmid in order?
ori - antibiotic resistance gene - promoter - MCS/GOI with His6 - terminator
How is the plasmid DNA purified from bacterial cells?
Restriction ligation - select for plasmid with antibiotics - centrifuge to seperate based on size - resuspension solution - lysis buffer - neutralization buffer - washes - elution buffer
What is in the resuspension solution?
TE buffer to resuspend cells, RNase to digest RNA
What is in the lysis buffer?
SDS to denature bacterial proteins, NaOH to denature chromosomal and plasmid DNA
What is in the first wash buffer?
High salt wash to remove residual contaminants
What is in the second wash buffer?
Ethyl alcohol to remove weakly bound molecules from Ni (are positive)
What is in the elution buffer?
10mM of Tris to release DNA from column
What is the equation for concentration? Units?
Absorbance/0.020 in µg/ml
What is the equation for total yield?
Concentration times total volume