Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.
Bio-Rad GMO Investigator Kit Genetically Modified Food, DNA Isolation, Electrophoresis, PCR, and Bioinformatics
Timeline • Thursday- Lecture, read and annotate protocol for HW, comprehension questions ,review concepts of PCR, Electrophoresis, DNA Isolation etc. as needed • Tuesday- Procedure quiz, extract DNA, isolate DNA, and run PCR • Thanksgiving! • Monday—debate/Volunteers pour gels (lab service) • Tuesday- Load samples, run gel, photograph gel and analyze for results, • Thursday- post lab quiz, Lab write-up due
Concept of Lab-DNA Isolation • Several steps are involved in extracting the DNA from the food samples since there is more than just DNA in the samples • The cellular membrane is broken open when the sample is ground up • The InstaGene™ matric binds to cations that assist enzymes in the sample that would degrade the DNA • When boiled the InstaGene™ matrix is activated, the cations bind to the matrix, and the heat inactivates the enzymes that would degrade the DNA • Centrifuging is a crucial step after boiling because the centerfuge pellets the remaining InstaGene™ matrix and the denatured bound enzymes • It is also important to not disturb the pellet when transferring the DNA lysate so that the InstaGene™ matrix and its componets don’t make it into the sample to be placed in the thermal cycler
Concepts of Lab-PCR • PCR • 94ºC Denaturing (1min)-During the denaturation, the two strands of the DNA template are melted apart to provide access for the PCR primers. • 59ºC Annealing (1min)- During the annealing step, the PCR primers recognize and bind to the DNA template. • 72ºC(2min) Extending/Elongating- Once the primers are bound, Taq DNA polymerase extends the primers to replicate the segment of DNA. • The PCR reaction will take approximately 3.5 hours to complete. • PCR Animation • http://www.bio-rad.com/LifeScience/jobs/2004/04-0522/04-0522_PV92_PCR.html
Concepts of Lab-Electrophoresis • Electrophoresis • The gel slab is porous and allow the DNA to flow through it • The larger the section of DNA is the slower it moves through the gel • The DNA is charged negatively so it runs to the positive electrode • The buffer the gel is submerged is conductive and allows the electricity to flow through the gel and stimulate the DNA • The conductivity of the buffer is due to a combination of CI- ions, NaOH, and a high pH
Lab Preparation GMO DNA Extraction (page 14) • Locate Mortar and pestle, clean • Add 500 μl of InstaGene matrix to each 16 screwcaptube • Set the water bath to 95–100°C at least 30 min before the lab
Lab Preparations GMO PCR (page 15-16) • Program thermocycler (page 65) • Get ICE, set-up 9 ice stations • Label • 8 screwcaps GMO + • Label 9 screwcap tubes "PMM" (plant master mix). • Label 9 screwcap tubes "GMM" (GMO master mix). • Thaw, aliquot MGO-positive DNA, store at -20C Perform this step 30 min–1 hr before the lab. Keep the tubes on ice. Thaw the master mix & primers and pulse-spin the tubes in a centrifuge to bring all contents to the bottom. Step 1—create master mix with primers • Add 550 μl of master mix to one PMM tube and one GMM tube. • Add 11 μl of green primers to the master mix in the PMM tube, and mix. Store on ice. • Add 11 μl of red primers to the master mix in the GMM tube, and mix. Store on ice. Step 3—aliquot master mix with primers to 8 student stations • Add 65 μl of the plant master mix with the newly added primers into each of the remaining 8 tubes labeled PMM. • Add 65 μl of the GMO master mix with the newly added primers into each of the remaining 8 tubes labeled GMM. • Put one PMM tube, one GMM tube, and one GMO (+) tube in an ice bath for each workstation
Lab Preparation GMO • Electrophoresis (page 17) Label • Label 8 flip-top tubes "LD" • Label 8 flip-top tubes "MWR” • Make gels—see page 18-19 (7 lanes, 3%, EtBr 1ul/50mL) Prepare molecular weight rule • Thaw the Orange G loading dye and PCR molecular weight ruler, and pulse-spin the tubes in a centrifuge to bring all contents to the bottom. • Add 40 μl of Orange G loading dye to the vial of PCR molecular weight ruler. Mix well and pulse-spin. • Add 25 μl of PCR molecular weight ruler to each of the 8 tubes marked "MWR". This can be prepared ahead of time and stored at 4°C for 1–2 months. Aliquot Loading dye • Add 70 μl of Orange G loading dye to each of the 8 tubes marked "LD". This can be prepared ahead of time and stored at 4°C for 1–2 months.
Lab Day 1 • Bring in a food item! (see page 26) • Fill your own breaker • Isolation-Part 1 • 1. Find your screwcap tubes and label one “non-GMO” and one “test”. • 2. Weigh out 0.5–2 g of certified non-GMO food and put it into the mortar. • 3. Add 5 ml of distilled water for every gram of food. To calculate the volumes of water you need, multiply the mass in grams of the food weighed out by 5 and add that many milliliters. Mass of food = ____ g x 5 = _____ ml • 4. Grind with pestle for at least 2 min to form a slurry. • 5. Add another 5 ml of distilled water for every gram of food. Mix or grind further with the pestle until the slurry is smooth enough to pipet. • 6. Pipet 50 μl of ground slurry to the screwcaptube containing 500 μlof InstaGenelabeled “non-GMO” using the 50 μl mark on a graduated pipet. Recap tube. • 7. Repeat steps 2–5 to prepare the test food sample. • 8. Pipet 50 μl of ground test food slurry to the screwcaptube labeled “test”. Recap tube. • 9. Shake or flick the non-GMO food and test food InstaGene tubes and place tubes in 95°C water bath for 5 min. • 10. Place tubes in a centrifuge in a balanced conformation and centrifuge for 5 min at max speed.
Lab Day 1 con. • PCR-Part 2 • 1. Number PCR tubes 1-6 but include group number. The numbers should correspond to the tube contents (example group 4: 4.1, 4.2, 4.3, etc.) • 2. Place each tube in a caplessmicrotubeadaptor and place in the foam float on ice. • 3. Referring to the table and using a fresh tip for each addition, add 20 μl of the indicated master mix to each PCR tube, cap tubes. • 4. Referring to the table and using a fresh tip for each tube, add 20 μl of the indicated DNA to each PCR tube, being sure to avoid the InstaGenepellet at the bottom of the tubes. Mix by pipetting gently up and down; recap tubes • 5. When instructed, place PCR tubes in thermal cycler.
Lab Day 2 • Electrophoresis • 1. Set up your gel electrophoresis apparatus as instructed. • 2. Obtain your PCR tube from the thermal cycler and place in the caplessmicrotubeadaptor. Pulse-spin the tube for ~3 seconds. • 3. Using a fresh tip each time, add 10 μlof Orange G loading dye (LD) to each sample and mix well • 4. Load 20 μl of the molecular weight ruler and 20 μl each sample into your gel in the order indicated in the packet • Load your molecular ruler in lane 7
Bibliography Biotechnology Explorer ™ GMO Investigator™ Kit http://www.ncbi.nlm.nih.gov/About/primer/bioinformatics.html