Lab 6 Enzyme Activity

Be able to identify independent and dependent variables.

• independent variable: on the x-axis, variables that the experimenter controls
during the experiment (time)
• dependent variable: on the y-axis, variable that is measured at the end of each
trial. (absorbance)

Be able to plot data and draw a best fit line

Be able to id the optimum temp or pH for an enzyme given data

• Optimum temp and pH is the one that is the highest on the graph
• The higher the substrate concentration the faster the enzyme activity, the lower
the substrate concentration the slower the enzyme activity.
• Enzymes reach max velocity because they can only work so fast to create product
from given reactant
• At low and high temp the enzyme is working very slowly,
o high temp because the substances are moving quickly therefore less
change of matching with the enzyme efficiently
o low temp the substance is moving slower lowering the chances of running
into the enzyme.
o pH’s that aren’t as high aren’t making as much product in the time
allotted.

Lab 7: Thin Layer Chromatography

Know how to obtain Rf on TLC

Rf = distance migrated by the pigment/distance migrated by the solvent front

Know the names of the 3 pigments we separated

• betacarotene or carotene (orange)

• Pheophytin (grey)
• Chlorophyll (dark green)
• Xanthophyll (yellow)
• Chlorophyll (light green)

Identify solvent, orgin, and solvent front on TLC

• Solvent front is the line the solvent moves to at the top (mobile phase)
• Orgin is where it starts
• solvent (developing solution ?) is the substance you set your TLC in.
• pigments that are most strongly attracted to the developing solutioin will move up
the chromatography plate fastest, pigments most strongly attracted to the silica gel
will move up the plate slower
Be able to identify the four stages of mitosis under the microscope
• metaphase, anaphase, telephase, prometaphase, prophase

Be able to determine if the cells are animal or plan in mitosis

Know that WL is wavelength and Abs is absorbance

Lab 8: Purification of GFP and BFP

Describe how a Sephadex (size exclusion) column works and that larger proteins come
off the column first.
• Smaller proteins pass through the pores between the beads
• Larger proteins flow on the sides of the column coming off first.

Be able to set a micropipettor to the correct setting and pipette liquid

Describe how PAGE electrophoresis works and that smaller proteins will travel faster.
• Denatured proteins will separate solely on molecular weight with larger molecular
weight proteins moving slower than smaller molecular weight proteins
• Proteins with more negatively charged R groups on amino acids will travel more
quickly from the cathode or negative end to the anode or postive end of the gel
• Smaller proteins will move more quickly through the gel than larger ones
• All proteins of similar size will travel equally far down the gel although these
proteins may not be the same protein
• A tightly folded and compact protein will travel more quickly through a gel than a
loosely folded one.

Identify the parts of PAGE:

• SDS: helps break up the bonds between the proteins, disrupting the secondary and
tertiary structure of them. It also binds to the proteins adding a heavier negative
charge than the proteins had before.
• mercaptoethanol: helps to break down bonds further breaking down the tertiary
structure of the proteins
• tracking dye: determine how far proteins have moved through the gel after
electrophoresis. runs faster than the proteins
• glycerol: add density to the protein sample and prevent it from floating away
from or out of the gel
• acrylamide: molecular base for the poly acrylamide gel that we use in
electrophoresis. The higher the concentration the smaller the pores of the gel are,
allowing analysis of
o larger molecules at low concentrations and
o smaller molecules at higher concentrations
• SDS and mercaptoethanol used to denatue proteins
• glycerol is in the buffer that increases the density of the protein sample

Know what is meant by protein markers and why we used them in the PAGE
 • Protein markers: mixture of proteins of known molecular weights are run along
side the unknown proteins. The molecular weights of unknown proteins can be
determined by comparing their migration distances to the migration distances of
the protein markers. You can use protein markers therefore to determine the
molecular weights of the unknown proteins.

Know what the graph in 8-3 was about.

• Graph the RF for each protein marker band vs the log of the marker proteins MW.
This ensures that the points will create a straight line. Has the actual distance
migrated vs MW been graphed instead the points would likely form a curve. Rf =
x-axis, Log MW on y-axis.
• y=mx+b where m is the slope and b is the y-intercept

Be able to use the equation for a straight line to get a value like we did in lab 8-3

Lab 9: Recombinant DNA Technology

Be able to draw and identify the 5 possible plasmid phage combinations

Only C contains recombinant DNA, all others contain non-recombinant DNA

QuickTimeª and a
decompressor
are needed to see this picture.
Be able to sketch how you spread the bacteria on a dish

Describe what is a restriction enzyme and why we used it in Lab 9

• Restriction enzymes are naturally occurring enzymes that cut DNA. Each
restriction enzyme cuts DNA only where a specific sequence of base pairs occurs
• The restriction enzyme EcoRI was used to cut the plasmid at a single point in the
lac Z gene

Define what is meant by recombinant and transformation

• recombinant: joining of DNA from 2 different sources in our lab the plasmid and
the phage
• transformation: exploiting the natural tendency of many bacteria to absorb
fragments of DNA from the surrounding medium Bacterial transformation is the
process by which bacterial cells take up naked DNA molecules.

Know why X-gal and ampicillin (antibiotic) were added to the agar in the Petri dishes
• One gene on the puc18 confers resistance to the antibiotic ampicillin. Bacteria
that contain this gene are able to grow in the presence of ampicillin, while
bacteria that lack this gene are not.
• The second lacZ gene codes for the enzyme B=galactosidase, used by E coli to
digest lactose
• When bacteria with the Lac Z gene are grown in a synthetic analog of lactose
called Xgal the enzyme digests Xgal and releases compound X which is blue
• the bacteria with a functional lacZ gene will produce a blue color in the presence
of Xgal
Whitefish Blastula
Onion Root Tip