Week+of+September+19

__September 19th__ The first thing we did today, was to test our skills at pipetting. The pipette that we will be using is the 20 microliters pipette. The most important thing to remember when pipetting is that the piston has two stops. In order to get the correct amount of fluid in the pipette is to only push until the first stop. The second stop in a "just in case" for if all of the liquid is not dispelled from pushing to the first stop. While practicing, we started by measuring larger amounts like 15 and 10 microliters. Then, we worked down to 1 and 2 microleters. (The following is the lab we did to practice pipetting.) The procedure for this part of the lab is as follows. (Taken from the CAFG Student Lab Manual) Materials Equipment 2-20μL Air displacement pipette (recommended: one instrument per 2 students) • Pipette tips • 12 Microcentrifuge tubes for 4 students • Plastic bag (1 bag per 4 students) • Permanent Marker (preferably black) Reagents Small beaker of water • Phenol Red Solution • Acid Solution • Base Solution • Assorted stock solutions of various reagents from //Genotyp //on instructor’s bench //. // Methods [...] 3. Practice changing the microliter volumes on the pipette. a. Change the volume of your pipette to 7.5 microliters (μL). b. Change the volume of your pipette to 12.0 microliters. 4. Practice adding liquid to the microcentrifuge tube. Figure 1.4. Pipetting Tips 8 a. Ready an empty microcentrifuge tube to practice mixing acids and bases. Every member of the group should practice on their own microcentrifuge tube. b. Set your pipette to 10.0 microliters. c. Draw in 10.0 microliters of yellow Phenol Red solution into a pipette tip. Observe the level of yellow solution in the pipette tip. There should be no bubbles in your tip. d. Expel the 10.0 microliters of Phenol Red into the bottom of the microcentrifuge tube. Observe the droplet on the bottom of the tube. It should be a yellow color. e. Eject your tip and put a new one on your pipette. You are now ready to add liquid from a new solution. f. Add 5.0 μL of Base solution to the tube, pipetting up and down carefully to mix the liquids. It should now be a fuchsia color. g. Change tips and add 10.0 μL of Acid solution to the tube. It should return to its original yellow color. Make sure to mix the solution until the entire solution is yellow. h. Change tips and add 15.0 μL of Base solution to the tube, it should return to its fuchsia color. 5. Practice removing liquid from the microcentrifuge tube. a. Set your pipette to 7.5 microliters. b. Touch the tip to the fuchsia liquid and draw in 7.5 microliters of liquid. c. Make sure you have not drawn in any air bubbles in the pipette tip. d. Eject the pipette tip. You are now trained to use an Air Displacement Pipette. 6. Assemble the reagents that you will use for your group of 4 students a. Number, initial, and label eight microcentrifuge tubes using a permanent marker according to the table on the right, and add the stated volumes from the stock solutions on the instructor’s bench to each tube. Be sure to carefully add the volumes to the bottom of the labeled microcentrifuge tube, because you will need almost every microliter for your group’s experiments. To add more than 20.0μL to a tube, simply set the pipette to 20.0μL and perform multiple transfers. b. Divide the tubes among the different members of your group. One member is responsible for getting an aliquot of one reagent for the entire group. At the end of this step, your group of four should have 8 tubes total, each for a different reagent. Your group will share these reagents in Chapter 2 and Chapter 3. 7. Store your reagents a. Close the lids of each tube tightly b. Gently put all of the tubes into a sealable plastic bag (such as a Ziploc® bag) c. Label the bag with initials of group members, and give it to your instructor to be placed
 * Change tips for every different reagent! **

After Practicing Pipetteing, we did the first step in the cloning, PCR (Polymerase Chain Reaction). The following is the instructions from the CAFG Student Manual: Lab 2: Polymerase Chain Reaction Purpose We will use Polymerase Chain Reaction to amplify the GFP gene. Using a DNA Polymerase called "Taq" and two primers that complement the two ends of the GFP DNA, we will create a huge number of linear copies of GFP DNA. In the next lab this DNA will be inserted into an antibiotic resistant vector. Materials Equipment 2-20μL Air displacement pipette (1 per 2 students) • Pipette Tips • 0.2mL (PCR) Tube • Thermal Cycler (1 per class) Reagents 5x PCR Buffer DNTPs GFP Forward Primer GFP Reverse Primer DNA Template • Taq Polymerase Methods You will be mixing together the appropriate reagents in given amounts, just as you practiced in the first laboratory session. Use the table on the right below to reference how much of each reagent, adding each reagent in from top to bottom in the order listed. It is important to remember good pipetting skills to ensure that there is enough of each reagent for everyone in your group.

Amount 5x PCR Buffer: 4.0μL DNTPs: 5.0μL GFP For.: 3.0μL GFP Rev.: 3.0μL DNA Template: 4.0μL Taq Polymerase:1.0μL Total: 20.0μL To see the effect of Taq later when running the gel, we used 1.0μL, 2.0μL, and 3.0μL in our PCR. When we finished adding all of the reagents to the tube, we put them in the PCR machine so that it can run through cycles of different temperatures. The high temperature breaks the dsDNA of the template allowing the primers to attach and anreplicate.

__September 21st__ Today, we did a Gel Electrophoresis. 1. Made a 1% Gel out of Agarose. 2. When the gel is made, it is put into a container filled with a liquid reagent mixed with Ethidium Bromide. 3. Then the ladder is inserted into the gel. 4. Next 2.0μL of the PCR product is loaded mixed with 10.0μL of Glycerol, a loading buffer. 5. Run the electricity for about 30 min. Because DNA is negatively charged it will flow through the gel and stop according to the size of the fragments of DNA. The target sequence is 1 kb. We are able to see the DNA because of the Ethidium Bromide dye. Results: After this entire process is over, photographed the gel to see if the PCR worked as planned. When we looked at the photograph, there was <range type="comment" id="682836">not any DNA in the gel. Now we must troubleshoot to see what went wrong in the PCR.
 * The gel is a neutral solution of acid and base.
 * 1% is a standard gel.
 * Higher percentage (denser) gel would be used for greater seperation between smaller segments of DNA
 * Ethidium Bromide is a chemical that latches onto the major grooves of DNA and causes it to be flourescent.
 * Ladder= gauge for the size of fragments of DNA.