1. Restriction Digests and Gel electrophoresis
DNA Technology
Restriction Digests and Gel electrophoresis

2. DNA Extraction Collect DNA sample (blood, saliva, hair follicle, skin)
open the cells using lysis buffer and a heat bath -2 components detergent to poke holes in membrane and proteinase K to cut apart the histones and free the DNA

3. Microsatellite regions
Almost all DNA between humans is identical (99.9%), except in non-protein coding sites called microsatellite regions
Where we look when comparing DNA to solve crimes or for paternity

4. DNA Amplification PCR=Polymerase chain reaction
Input: nucleotides, taq polymerase (like our DNA polymerase but won’t denature with heat), and primers for region we want to copy.
Cycles of heat to unzip DNA, temperature for primers to bond, and temperature for taq polymerase to add nucleotides-See animation

5. Restriction Digest Now cut up the DNA based on its base pair sequence
We use restriction enzymes from bacteria. They cut at very specific sequences. Example: BsuRI GGCC going 5’ to 3’
Why would bacteria need enzymes to cut up DNA?

6. Practice
Where would BsuRI cut for the following individuals and how many pieces would result?
Person 1 5’TGGCCATGGCGGCC3’
3’ACCGGTACCGCCGG5’
Person 2 5’TTCCGGCCTCCAGA3’
3’AAGGCCGGAGGTCT5’

7. Volume We will be measuring tiny volumes-microliters written µL.
There are 1000 µL in I mL. There are 1000mL in 1 L. So 1 µL is 1,000,000 of a liter=very small.
We use micropipettes
Use p20- can measure between 2 and 20 µL accurately
Top number = tens place, middle= ones place, and bottom=tenths place

8. Micropipettes Very expensive-$300 each
Liquid must never touch the barrel, it must always be in a new tip
Always hold the micropipet
vertically
Work at eye level

9. Practice volumes How would you dial to 6.5 µL?

10. Diagram
Draw pipette diagram on board

11. Micropipet Use 1. Dial to correct volume and lock plunger
2. Put on fresh tip
3. Push plunger down until first stop=resistance
4. Insert tip into liquid and release plunger SLOWLY
5. Place tip into container to move liquid to
6. Push down to the 2nd stop. Remove micropipet BEFORE you release plunger

12. Gel Electrophoresis A process used to separate our cut DNA by LENGTH!
DNA is NEGATIVE
What attracts a negative?
Negative at the top of the gel, positive at the bottom
Electric current pulls DNA
through the gel
Which pieces will move faster if
it is like the game?

13. Activity
The restriction enzyme you will use will cut at 5’ to 3’ GG CC, between the Gs and Cs
Draw where you would cut the sequences and draw a line where they would move to on the gel
Which person committed the crime?