Big Idea 3 – Investigation (Lab) 9
We did a variation of this lab at the Cold Spring Harbor DNA Learning Center West…
The next few slides highlight the differences between what you did and what this lab specifically
asked that you do…which is to pretend that you are working a crime scene, have a DNA sample, and
will cut with restriction enzymes to look for restriction fragment length polymorphisms (RFLP’S)
Big Idea 3 – Investigation (Lab) 9
Crime scene DNA
Suspect DNA
Restriction fragment length
polymorphisms (RFLP’s = “rif lips”)
The differences in restriction sites
found on homologous chromosomes
giving rise to different numbers and
lengths of restriction
fragments...review chapter 20
powerpoint for more detail.
Amplified section of the
Amplified section of the
DNA from the crime scene same DNA segment from the
Big Idea 3 – Investigation (Lab) 9
IDEAL GEL
A gel showing lambda phage DNA (same DNA you
cut at the DNA learning center) cut with three
different restrictions enzymes and an uncut
negative control.
Look at chapter 20 for a review of how gel
electrophoresis works…
Big Idea 3 – Investigation (Lab) 9
Calculating the Standard Curve
Big Idea 3 – Investigation (Lab) 9
Calculating the Standard Curve
2.3
2.3
3.0
3.5
4.2
5.8
6.3
Big Idea 3 – Investigation (Lab) 9
Calculating the Standard Curve
Size of fragment (bp)
Relationship between DNA fragment size in bp
and migration distance in a 0.8% agarose gel.
25000
2.3
2.3
3.0
3.5
4.2
5.8
6.3
20000
15000
10000
5000
0
0
2
4
6
8
Migration distance (cm)
There appears to be an exponential relationship…
Or…small fragments move exponentially quicker than
larger fragments…
If so, then using a log scale on the y-axis should result
in a linear relationship…(next slide)
Big Idea 3 – Investigation (Lab) 9
Calculating the Standard Curve
100000
10000
1000
100
2.3
2.3
3.0
3.5
4.2
5.8
6.3
10
1
0
2
4
6
8
20000
R2=0.93057
2000
0
2
4
6
8
Big Idea 3 – Investigation (Lab) 9
Calculating the Standard Curve
20000
R2=0.93057
2.3
2.3
3.0
3.5
4.2
5.8
6.3
2000
0
2
4
6
8
This is now our standard curve…we can use it to do
what?
To calculate the sizes of the BamHI and EcoRI
bands…
Simply measure the distance traveled by the
band and then use the curve to determine the
size.
Big Idea 3 – Investigation (Lab) 9
Calculating Size of Unknown Bands
20000
14,000
2.3
2.3
3.0
3.5
4.2
5.8
6.3
2.5
R2=0.93057
14,000
2000
0
2
4
6
8
Calculate the size of the largest fragment for the
BamHI digest.
The migration distance is 2.5 cm.
Therefore, the size is approximately 14,000 bp
(I actually used the formula for the line,
which excel can give you, instead of the graph
itself, but the AP will require you to use the
graph).
Big Idea 3 – Investigation (Lab) 9