Primers/ Primer Design
What is 19-22 nucleotides?
Chemical used to induce expression of Taq
What is IPTG?
The purpose of Tris buffer
What is to maintain pH?
"Smelly" reducing agent added to loading buffer to reduce disulfide bonds
What is beta-mercaptoethanol?
This is the first thing you try if you see nonspecific amplicons in a PCR reaction
What is increase annealing temperatures?
Optimal primer melting temperature
What is 60°C ?
IPTG is a structural analog of
What is lactose (or allolactose)?
The purpose of heating our protein sample at 75°C for 1 hour
What is to denature heat-labile E. coli proteins?
Approximate size of Taq protein
What is 96 kDa?
The reason PCR works better on shorter DNA than longer DNA
What is polymerase processivity (polymerase more likely to remain associated with DNA template)
Only part of the primer that must bind template for PCR to work
What is the 3' end?
What the acronym Taq stands for
Enzyme, often added to lysis buffer, that destroys the cell wall of bacteria
What is lysozyme?
Purpose of running standards on gel
What is to estimate size of proteins in the lysate?
We chose AfeI to digest our pTAQ plasmid because
What is there were 2 sites within the Taq gene insert?
When calculating primer melting temperature, G's and C's are worth
What is 4°C?
IPTG is used instead of lactose to induce protein expression because
What is bacteria cannot metabolize IPTG?
Name given to supernatant after lysis
What is lysate?
Protein we used to create our standard curve during the Bradford Assay
What is bovine serum albumin (BSA)?
Two conditions of PCR that are most often modified to optimize the reaction
What is annealing temperature and extension time?
Formula used to determine how often (on average) a primer could bind to a random length of DNA
What is 4n? (where n is the length of the primer)
The purpose of EDTA in the protein lysis buffer
What is to chelate Ca2+ ions and thereby inhibit proteases?
Reason for using polyacrylamide to separate proteins instead of agarose (like for DNA)
What is proteins are way smaller than DNA? -or- What is agarose can't make small enough pores?
Detergent added to proteins to denature them and give them a negative charge
What is SDS (sodium dodecyl sulfate)?
Definition of a unit of restriction enzyme
What is the amount of enzyme that will cut 1μg of DNA in 1 hour?