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

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

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

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

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

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

Cathode (BLACK) Wells Anode (RED) MOVING DIRECTION

- + 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).

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.

Day Two

Polymerase Chain Reaction

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

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

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

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

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

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

Loading the Gel

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.

Well Loading Demonstration

Gel Electrophoresis

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

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

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

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?