Name
LAB REPORT 1: DNA Characterization
40 points
Experiments 1 and 2: Isolation of DNA and UV Spectrophotometry
1
(30) Complete the table below. The DNA control is the DNA you extracted in Experiment 1.
NanoDrop
Program
Sample
A260
A280
Concentration
A260/A280
DNA Control
BSA @ 100
BSA @ 10-1
Nucleic Acid
BSA @ 10-2
BSA @ 10-3
BSA @ 100
BSA @ 10-1
Protein
BSA @ 10-2
BSA @ 10-3
DNA + BSA @
100
DNA + BSA @
10-1
DNA + BSA @
10-2
DNA + BSA @
10-3
2
Nucleic Acid
(20) Generate a set of five graphs, each showing BOTH the DNA and BSA data generated from
the Nucleic Acid program.
1 Unmixed DNA and unmixed BSA @ 100
2 DNA + BSA @ 100
3 DNA + BSA @ 10-1
4 DNA + BSA @ 10-2
5 DNA + BSA @ 10-3
You can trace the graphs in order to superimpose them.
3
(10) What is the target A260/A280 ratio when determining DNA purity? Why is this the desired number? Why does
this ratio decrease with increasing impurity?
4
(5) Assume that you have a DNA stock that is 75 g/ml. Use the formulas on page 46 of the lab manual to
mathematically demonstrate that single-stranded DNA absorbs more UV than double-stranded DNA
Experiment 3: Purification of DNA and UV Spectrophotometry
5
(10) Attach a copy of YOUR chromatogram
6
(10) Using either your own data or the data posted online (specify which) calculate the Rf values for each base
Base
Distance from Origin
Rf Value
Adenine
Thymine
Cytosine
Guanine
Solvent Front
Experiment 4: DNA Sequence Specificity
7
(10) Attach a copy of your gel.
8
(10) Briefly describe the premise of this experiment. What are we attempting to determine? Why did we set up the
experiment in the manner that we did?
9
(20) Complete the table using information in Appendix IX or the New England Biolabs catalog.
Enzyme
EcoRI
BamHI
XbaI
HindIII
# Units
per rxn
Expected Site
Frequency
Expected Number
of Sites
Actual Number
of Sites From
Gel
Actual Number
of Sites From
Appendix IX
10
(5) Did your observed results match your expectations? Explain why/why not.
11
(5) What is your expectation regarding the distribution of fragments from each digestion? Why?
12
(5) Did the distribution of fragments match your expectation? Explain why/why not.
13
(20) Use the data in Appendix IX to place the restriction sites on the map below. The same data table is also
located in the NEB catalog
Grade = Total Points x 40 points
160