2. Warm-Up Go over today’s procedure with your table members
I will come around and check your pre-Lab
Video Notes
Materials list
Written lab procedures

3. SAFETY PROCEDURES WEAR YOUR GOGGLES AT ALL TIMES!
(Don’t Worry! We will be Dorky TOGETHER!!!!)
2. DO NOT EAT FOOD DURING THE LAB
3. DO NOT CONSUME ANY LAB MATERIALS AND EQUIPMENTS

4. Lab Procedures WEAR YOUR GOGGLES AT ALL TIMES!!!!!
Remove your digested DNA samples from the refrigerator
Tap the tubes on the table top to bring down all the liquid into the bottom of the tube
Using NEW tips each time, add 5μL of the loading dye (marked as LD) and gently mix the dye and your DNA sample

5. Lab Procedures – Loading the gel
Load the agarose gel onto the electrophoresis apparatus
Fill the electrophoresis chamber with 1x TAE buffer
make sure that the wells are near the black (-) and the bottom of the gel is near the red (+)
Load each of the samples into the wells of the gel

6. Lab Procedures – Loading the gel
Lane 1 = S, DNA size standard 10 μL
Lane 2 = CS, green tube 20 μL
Lane 3 = S1, blue tube 20 μL
Lane 4 = S2, orange tube 20 μL
Lane 5 = S3, violet tube 20 μL
Lane 6 = S4, red tube μL
Lane 7 = S5, yellow tube 20 μL

7. Lab Procedures – Gel Electrophoresis
Place the lid on the electrophoresis chamber
The red and black jacks on the lid of the electrophoresis chambers will match with red and black jacks on the base
Red  Red
Black  Black
Turn on the power at 100V for 30 minutes

8. Cathode (BLACK)
Wells
Anode (RED)
MOVING DIRECTION

9. - + Cathode Anode Buffer Power Supply Dyes Agarose gel
During electrophoresis, water is electrolyzed which generates protons (H+ ions)at the anode (positive) and hydroxyl ions (OH -1)at the cathode (negative). The cathode (negative) end of the electrophoresis chamber then becomes basic and the anode (positive) end becomes acidic.
The electrode at which electrons enter the gel box from the power supply (along the black wire) is called the cathode and is negative (-). The electrode at which electrons leave the box and re-enter the power supply (along the red wire) is called the anode and carries a positive charge (+). The flow of electrons sets up a potential energy difference between the electrodes. This is known as potential, and is measured in volts. It establishes an electric field through which the ions in the gel box fluid migrate. The migration of ions in the fluid creates electrical current which is measured in milliamperes (milliamps).

10. Running the Gel
Place the cover on the electrophoresis chamber, connecting the electrical leads. Connect the electrical leads to the power supply. Be sure the leads are attached correctly - DNA migrates toward the anode (red). When the power is turned on, bubbles should form on the electrodes in the electrophoresis chamber.

11. Day Two

12. Polymerase Chain Reaction

13. The Central Dogma of Biology
Go thru central dogma
Include gene in DNA
Appllies universally to all living organisms
Yet so malleable we have diverse life living in almost every available niche on our planet
In our workshop, we will focus on content and techniques that will take us through this central dogma
Today we’ll start with an introduction to the structure of DNA and how it replicates

14. DNA Replication and DNA Polymerase
DNA has to be replicated when cells divide to make new cells.
Examples?
The entire genome must be uncoiled and replicated exactly. It is a very complicated process I am going to go over briefly because I want you to learn about the most important aspect of it: DNA polymerase
Replication starts at multiple sites along the DNA molecule called origins of replication
At the ORI, DNA gyrase removes the supercoils and DNA helicase untwists the double helix and separates the left and right sides of the ladder
This creates a Y-shaped region of DNA called the replcation fork
Then, DNA polymerase attaches with several other factors and synthesizes a new matching strand.
\DNA polymerase always goes 5 to 3 prime – show in diagram so on one strand it can move straight along and this is called the leading strand
On the other it does several segments of 5 to 3 prime on the lagging strand and those segments are called Okazaki fragments which are connected together by DNA ligase

15. Polymerase Chain Reaction - PCR
PCR amplifies DNA
Makes lots and lots of copies of a few copies of D NA
Can copy different lengths of DNA, doesn’t have to copy the whole length of a DNA molecule
One gene
Several genes
Lots of genes
PCR = Artificial process which imitates natural DNA replication

16. What do we need? DNA sample which you want to amplify DNA polymerase
Nucleotides
Called dNTPs
(A’s, T’s, C’s, G’s)
Pair of primers
One primer binds to the 5’ end of one of the DN A strands
The other primer binds to the 3’ end of the anti- parallel DNA strand

17. How PCR Works Put all reagents into a PCR tube
Break the DNA ladder down the middle to crea te two strands, a 5’ to 3’ strand and a 3’ to 5’ stra nd
Melting or heat denaturation
Bind each primer to its appropriate strand
5’ primer to the 5’ to 3’ strand
3’ primer to the 3’ to 5’ strand
Annealing
Copy each strand
DNA polymerase
Extending

18. How PCR Works Denaturation: Annealiation : Extension:
~ 95ºC for 30 seconds
Annealiation :
~ 50ºC for 30 seconds
Extension:
~ 72ºC for 1 minute
30-35 cycles

19. Loading the Gel

20. Loading the Gel
Carefully place the pipette tip over a well and gently expel the sample. The sample should sink into the well. Be careful not to puncture the gel with the pipette tip.

21. Well Loading Demonstration

22. Gel Electrophoresis

23. Gel Electrophoresis of DNA
Gel electrophoresis detects the number of nucleotides in a fragment of DNA
e.g., the number of nucleotides in a DNA re gion which was amplified by PCR
Can be used to tentatively identify a gene b ecause we know the number of nucleotides in many genes

24. Agarose Electrophoresis Loading
• Electrical current carries negatively-charged DNA through gel towards positive anode (red) electrode
Buffer
Dyes
Agarose gel

25. Agarose Electrophoresis Running
• Agarose gel lines up DNA fragments according to size – Small fragments move farther than large fragments. Why?
Gel running

26. Exit Ticket Questions
What would happen if we did not use the restriction enzyme before running the gel?
What would happen if we loaded the DNA on the positive side of the machine?