Untitled Flashcards Set

BCOR 1400 Lab Practical Study Guide TA: Kate Zoller

Lab 1: Lab Skills

Know how to pipette accurately

To aspirate liquid, push the plunger down to first stop, put the pipette tip into the liquid, slowly release the plunger to aspirate the liquid

To dispense liquid, push plunger down to the first stop (all or most of the liquid should leave the pipette tip). Continue pushing plunger to the second stop

Lab 2: Protein Structure

Primary structure

Secondary structure

Tertiary structure

Quaternary structure

N- and C- termini

Active site – Where on the enzyme is it? What happens here?

Where in the protein are the hydrophobic and hydrophilic amino acids?

Alpha helices and beta pleated sheet – Can you point out which parts of the protein are alpha helices and beta sheets?

Lab 3: Protein Purification

Understand the concept of ion-exchange chromatography

Steps:

1. Equilibrate the resin in the column by adding an equilibration buffer.

In our case, the equilibration buffer was 25 mM glycine (pH 9.2).

Q: What proteins would you expect to elute from the column upon adding the equilibration buffer?

A: None. You haven’t added any protein to the column yet, so no protein would elute at this step (trick question)

2. Add the solution containing your protein of interest to the column and collect the flow through.

In our case the solution we added to the column was egg white diluted in 25 mM glycine buffer (pH 9.2).

Q: What charges would you expect the proteins that elute at this step to have?

A: Negative (repelled from negatively charged Sephadex beads) and neutral (not attracted to negatively charged Sephadex beads) proteins would elute at this step.

3. Wash out unbound proteins from the column using the equilibration buffer.

In our case the equilibration buffer was 25 mM glycine at pH 9.2.

Q: What charge(s) would you expect the proteins that elute at this

step to have?

A: Negative (repelled from negatively charged Sephadex beads) and neutral (not attracted to negatively charged Sephadex beads) proteins would elute at this step.

4. Wash out weakly unbound proteins from the column using a low salt solution.

In our case the low salt solution was 100 mM NaCl in equilibration buffer (i.e., 25 mM glycine (pH 9.2)).

Q: What charge(s) would you expect the proteins that elute at this

step to have?

A: Weakly positive

5. Elute your protein of interest using a high salt solution

In our case the high salt solution was 750 mM NaCl in equilibration buffer (i.e., 25 mM glycine (pH 9.2)).

Q. What charge(s) would you expect the proteins that elute at this

step to have?

A: Strongly positive

Lab 4: Enzyme activity

Terms:

Active site

Substrate

Activation energy

Lab 5: Determining Protein Concentration

Be able to determine the concentration of protein in an unknown solution using a standard curve

Q: You generate a standard curve of BSA using a Bradford assay. The

equation of the curve is: y=0.02x (y-axis is absorbance, x-axis is ug of

BSA). You measure the absorbance of 100 uL of an unknown solution,

and the spectrophotometer reads 0.6 AU. What is the concentration of protein in the unknown solution?

A: 0.3 ug/uL

Be able to calculation the volume of sample needed for loading an SDS PAGE well

Q: How many uL of unknown sample will you need to load 15 ug of

protein in an SDS-PAGE well? The concentration of protein in the

unknown sample is 2 ug/uL

A: 7.5 uL

Lab 6: SDS-PAGE

Know what SDS PAGE is used for

Which proteins move faster through an SDS PAGE

What direction do all proteins migrate in an SDS PAGE? (from negative to positive)

Lab 7: Bioinformatics and PCR

Terms:

PCR (polymerase chain reaction)

Q: What is PCR used for?

A: Amplifying a specific target sequence of DNA determined by the primers used. (i.e., Making lots of copies of DNA. The sequence that is copied is the region flanked by the forward and reverse primers.)

Q: What are the necessary components of PCR?

A: primers, polymerase, nucleotides, template DNA

Primers (forward and reverse)

DNA template

Polymerase

Nucleotides

Agarose gel

Q: What are agarose gels used for?

A: Separating DNA (or RNA) by size

Q: Toward which electrode (+ or -) does DNA (or RNA) run on an agarose gel?

A: Towards the positive electrode because DNA (and RNA) is negatively charged.

Q: Do small or large DNA (or RNA) fragments move faster through an agarose gel?

A: Small DNA (or RNA) fragments move faster (large fragments move slower)

Restriction enzymes

Be able to do the restriction enzymes portion of the Bioinformatics worksheet on your own, including the agarose gel portion

Lab 10: C. elegans

Terms:

Hermaphrodite

Chemotaxis

Model organism

Wildtype

Mutant