Exam #1 Review

By: Morgan Homan

 Practical Exam
-Be able to do these
• Scale & Pipette

• Serial Dilutions & Standard Curve (1:2 dilution)

• -Identify different laboratory tools

• -Spectrophotometry of DNA

• -Loading a gel

• -Affinity Chorography
 Scale & Pipette
How to tare a scale

How to read the scale correctly

How to read all three pipettes

Note: your lab manual is a great review source

Serial Dilutions & Standard
Curve
Review how to do a (1:2) serial dilution

*** remember to throw out the last 100 microliters

from the 2nd to last well

Every well should have a total of 100 microliters at

the end of your dilution

The blank should be your last well

Identify laboratory tools
Pipette (which range?) • Roller pipette
• Centrifuge
Spectrophotometer • Microcentrifuge tubes
Blue/yellow tips • Heat block
• Gloves
Test tube rack • Glass tubes
• Cuvettes
Scale
Spectrophotometry of
DNA
Know the different wavelengths, especially which
wavelengths DNA and Protein absorb the most

Remember that absorbance is directly related to

concentration

Know how to blank the machine

 Gel Loading Skillz
Step one: Breath. It’s not that bad. Just go slow.
Steady your hand with the other hand on your wrist, if
necessary
Go all the way into the well before pumping out the liquid—
don’t lose all your sample because it floated away over the top
of your well.
Inject sample SLOWLY—go too fast and it will mushroom out
DON’T stab through the gel. This will lose you points.
Don’t go to the second stop—don’t want bubbles
Make sure to not release the plunger until you pull it out of the
gel, or you will draw your sample back up accidentally. Practice
the motion, holding your thumb down until you are out of well.
 Affinity
Chromatography
Review the steps to affinity chromatography, pay
attention to the specific vocabulary like the different
buffers, what ‘elution’ means, and what the ‘flow
through’ is.
 Written Exam
-Take in the testing center anytime during the test week
 Reading a Gel
Know how to read band size compared to a ladder

Understand where longest/shortest fragments

appear

Know the mechanics of DNA migration—why does it

move?
 DNA fingerprinting
Understand what a restriction endonuclease is, and how
we use them

Know the possible applications: crime scene

investigation, species identification, paternity
determination, ancestry identification, creating
evolutionary trees… In each case, the ability to compare
individuals based on DNA sequence gives important
information. For example, a crime suspect may have the
same DNA fingerprint as a sample from the crime scene.
 ELISA
What are we testing for? –antigens…which are?
– control will not have antigen ( will be clear)
+ control will have antigen ( will be colored)
1st add primary antibody (binds to antigen of interest) : If it
doesn’t bind then that means that there is no antigen to
bind to.
2nd add secondary antibody (binds to primary antibody):
HRP enzyme is already on the secondary antibody
HRP enzyme: Converts the substrate into a visible color for
detection (HRP enzyme + substrate = color)
3rd add the Enzyme substrate (converted by the HRP
enzyme into a visible color for detection)
ELISA (cont.)
 Affinity Chromatography
What is the purpose? –Isolate/purify a protein
Know what a ligand is. Just do it.
NOTE: Ligand is bound to matrix—never comes out in
any flow-through
Understand what comes out in each flow-throw—
protein of interest does NOT come out on first flow-
through. That would utterly defeat the point and
probably destroy the universe.
Second step: wash the column (get stuck things off)
Third step: elute the column (remove protein of
interest)
Understand: contamination, size of protein in band (where
are the big/small bands?), How pure is the sample?
Affinity Chromatography
(cont.)
(*Don’t worry about desalting)
 DNA Quantification
SDS denatures proteins and biological membranes,
and adds overall negative charge
Protease K destroys proteins, allows DNA to be
broken free from cell

EDTA chelates divalent metal cations (Mg2+) and

thus inhibits DNAses from destroying your DNA. Very
important.

SDS and protease K can also help inhibit DNAses.

 [DNA] and %DNA
Wavelength Absorbance
DNA was extracted from a 600 mg sample of
A260 .70 fish and allowed to air dry. It was then
A280 .2 resuspended in 400 µl TE buffer. A 1:5
A234 .34 dilution of the resuspended DNA- was made
and then analyzed in the spectrophotometer.
A320 .019
Discuss the purity of this sample using ALL of What is the concentration of DNA in the
the wavelengths tested. resuspended sample?
A260 * (known [DNA]) * Dil. Factor = [DNA,µg/ml]
A260/A280 = 0.70/0.20 =3.5– Evidence for 0.7 * 50 μg/ml * 5 = 175 μg/ml
clean DNA-no proteins (<1.7)
A234/A260 = 0.34/0.70 =0.485– Evidence for What is the percent of DNA in Fish tissue?
clean DNA-also no proteins (>.5)
A320/A260 = 0.019/0.70 =.027– The cuvette [DNA, µg/ml] * (resuspension TE) = amount DNA, µg
was clean and free from particulates (>.05) 175 μg/ml * 0.4ml = 70μg
Amount DNA/amount tissue * 100 = %DNA
*****DNA: there is a known standard for 70 μg _ * 100% = 0.0116%
A260 (absorbance value). A reading of 600,000 μg
1.0A at A260 = the DNA is 50ug/ml
 DNA Quantification
(cont.)
REMEMBER TO ACCOUNT FOR ALL THE
FLUID, BLAST IT!
But really, we don’t give a rat’s fanny
about anything except the DNA—so make
sure you account for liquid used to
suspend, and any dilutions. That’s all the
math is doing, anyway. Review that. It
might help you remember why you’re
multiplying/dividing where and when you
are.
Protein Quantification
We us the spectrometer to find the amount of
protein present, just like DNA—but different
wavelengths.
The Dye (coomassie brilliant blue) attaches to the
protein (at free NH3+), and the dye/protein
complex gets shifted to a higher absorbance value
(465 to 595). –this means we can read it without
worrying about other things absorbing light at that
higher wavelength (because hardly anything else
in your solution will absorb that frequency).
 Protein Quant. Example
A Protein Quantification experiment was Tube # (X) A 595 Reading (Y)
performed similar to the one you did in the
laboratory. 0 µg 0
20 µg BSA 0.080
0.3 g of a powder was dissolved in PBS to
yield a total volume of 5 ml. Since the protein 40 µg BSA 0.150
was dissolved together with water to total
five milliliters of volume, you will not need to 50 µg BSA 0.175
account for a volume change due to the Sample replicate 1 0.160
powder.
Sample replicate 2 0.165
The dissolved protein sample was diluted 1:10.
X*X X*Y
Two duplicate samples were prepared for
0 0
quantification using 20 µl of diluted sample
protein, 75 µl of PBS and 5 ml of Coomassie 400 1.6
Blue Dye.
1600 6
2500 8.75
Determine Slope: = .00363 Total= 4500 Total= 16.35
∑(X*Y)/∑(X*X) total= Slope = .00363
.00363
 Example (cont.)
1. Amount of protein as measured in the
cuvette :
Y=mX
(Use Average reading for Y --add both sample
readings up and divide by 2) Y = 0.1625
OD = m * µg
0.1625 = 0.00363 * x
x = 44.76 µg
3. Concentration of protein in the original
dissolved sample (in the 5 ml sample):
(Multiply the diluted sample concentration
by the dilution factor)
2.238µg/µl *10 = 22.38g/l
2. Concentration of protein in the 1:10 diluted
sample:
(divide the amount in the sample by the
volume of the sample)
µg / volume
44.76 µg / 20 µl = 2.238µg/µl
4. Determine the amount of protein present
in the 0.3 g powder sample:
Convert the units to be compatible with the
total volume in ml:
22.38 µg/µl = 22.38mg/ml
Then multiply by 5ml to cancel out the fluid
and be left with amount of solid in mg:
22.38mg/ml * 5ml = 111.9 mg
 Info from your packets:
What is SDS PAGE? Why the proteins
Why is protein concentration by move? What determines how proteins
spectrophotometry harder than move in the gel?
determining DNA concentration? Which
one requires a standard curve, and why? Proteins are first denatured by exposure
to SDS. SDS denatures proteins by
Since proteins are not made of a competing for hydrophobic bonds and
uniform mixture of amino acids a also gives the protein a uniform negative
constant relationship between protein charge. Adds a uniform negative charge.
concentration and absorbance at a (without the – charge it wouldn’t do
specific wavelength does not exist. Thus anything. It wouldn’t move) (DNA
proteins standards must be used with doesn’t need SDS because the DNA
some means of measuring the amount already has a – charge.)
of protein like coomassie blue.
The denatured protein is then placed in
A constant relationship does exist for an electric field and migrates towards
absorbance of DNA at 260nm, thus a the positive pole. Larger proteins take
standard curve is not necessary if the longer to make their way through the
DNA is of high enough purity. pores of the gel, while smaller protein
make their way more easily.