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)

Restriction fragment length polymorphisms (RFLP’s = “rif lips”)

Amplified section of the DNA from the crime scene

Amplified section of the same DNA segment from the suspect.

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.

Crime scene DNA Suspect DNA

Big Idea 3 – Investigation (Lab) 9

Big Idea 3 – Investigation (Lab) 9

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.

IDEAL GEL

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

2.32.33.03.54.25.86.3

Big Idea 3 – Investigation (Lab) 9

Calculating the Standard Curve

2.32.33.03.54.25.86.3

2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 70

5000

10000

15000

20000

25000

Migration distance (cm)Si

ze o

f fra

gmen

t (bp

)

Relationship between DNA fragment size in bp and migration distance in a 0.8% agarose gel.

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

2.32.33.03.54.25.86.3

2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 71000

10000

100000

2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 72000

20000

R2=0.93057

Big Idea 3 – Investigation (Lab) 9

Calculating the Standard Curve

2.32.33.03.54.25.86.3

2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 72000

20000

R2=0.93057

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

2.32.33.03.54.25.86.3

2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 72000

20000

R2=0.93057

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).

14,000

2.5 14,000

Big Idea 3 – Investigation (Lab) 9