Detecting Genetically Modified Foods by PCR Carolina Kit

Timeline Monday—Lecture, volunteer aliquot Tuesday—procedures quiz, isolate DNA cells, amplify DNA (PCR) Wednesday—Volunteer pour gels Thursday—load samples, run gel, photograph gel Monday & Tuesday--Bioinformatics Thursday—Lab write-up due

Write-up Annotate handout Data draw a gel and mark each banding site, staple picture to lab that you turn in to me Results and Discussion—answer all parts Bioinformatics—complete worksheet

Background Information Use website o.html o.html Review Protocol Section: introduction methods bioinformatics Animations—How did these foods become GM? Cutting and pasting A & B Transferring and storing A & B

Prep. For lab—GM foods 2-3 weeks before 1. Buy soy or corn food product (you may bring something you want to test) Example: corn chips, artificial bacon bits, corn muffin mix, granola, energy bars, protein powder, pet food 2. label tubes 3. Make more TAE buffer Monday—Aliquoting 1.soy beans—wild type 2.Put 1 round-up ready piece in a test tube for each group testing (4 tubes) uL (1mL) of Edward’s buffer (16 tubes) uL of Isopropanol (16 tubes) uL of TE/RNase A (16 tubes) uL of 35S primer/loading dye (16 tubes) uL of Tubulin primer/loading dye (16 tubes) 8.20 uL of marker/ladder pBR322/BstNI (8 tubes) Tuesday— get ice Wednesday—Pour Gels Pour 2% gels, add ethidium bromide (200ng/mL final or 1uL of 10mg/mL stock in gel prepared for 50mL), 6 well comb, TAE buffer, 8 gels (8 grams agarose add up to 400mL TBE buffer) Prepare UV trans. and camera

Preparing gels ___ grams agarose Add up to ___mL buffer Melt in microwave, let cool Set up trays—use 6 well comb Add 1uL ethidium bromide Pour about 30-50mL into each tray

Each Group will isolate DNA from 2 samples 1. Wild type=wt (groups 1-4) Negative control OR Round-up ready=RR (groups 5-8) positive control 2. Food item (you bring this in)=F

Lab Day 1 Part II: isolate DNA Set-up a boiling water bath ASAP, use the floats at station 9 to hold your test tube at the top (hot plate, beaker, foil)—use foil on top with holes or floats Plants not growing—use embryo Round-up Ready leaf will be in a test tube for you already Label tubes--be clear on what food item you are testing and what plants you are testing Smash with pestle to break cell walls of plants and foods Edward’s buffer is used to dissolve lipids that compose the cell membrane, boiling denatures proteins, DNA then floats to top Isopropanol (alcohol) will cause nucleic acids to collect on outside of tube Make sure to face hinge out, so you know where to look for the nucleic acids TE/RNase A— dissolves the nucleic acid pellet (TE=tris-EDTA), Tris provides a constant pH of 8.0, EDTA binds cations required for DNase activity, RNase A digests RNA, which composes the vast majority of nucleic acids After step 18, keep DNA on ice as you set-up your PCR reaction NO Mineral oil I will store your PCR samples in the freezer after PCR

Each Group will PCR 4 samples Test for 35S promoter—35S promoter of cauliflower mosaic virus (cmV) and detects GM promoter 1. Wild type=wt (groups 1-4) or Round-up ready=RR (groups 5-8) 2. Food item A (you bring this in)=FA Test for Tubulin—+ control for plant DNA 3. Wild type=wt (groups 1-4) or Round-up ready=RR (groups 5-8) 4. Food item A (you bring this in)=FA

Lab Day 1 Part III:PCR 4 PCR tubes per group NO Mineral oil I will store your PCR samples in the freezer after PCR Add 35S primer/loading dye mix to PCR tubes, then add DNA (do twice) Add Tubulin primer/loading dye mix to PCR tubes, then add DNA (do twice) Label each PCR tube (4) with this number of your group and the sample number (example: group 2, 3 test tube would be 2.3 Store on ice until I run PCR machine

Lab Day 2 Part C: electrophoresis photograph Skip set 10, we already added ethidium bromide Loading dye already put in with primers, so you can just load pBR322/BstNI 1857 bp, 1058bp, 929 bp, 383 bp, 121bp

Gel loading—change in protocol 1.Marker 2.Tubulin (wt or rr) 3.35S (wt or rr) 4.Tubulin food 5.35S food 6.empty Make sure to record what is in each lane in your lab notebook

Bioinformatics Follow the directions in lab Complete the worksheet

results Band sizes ?bp 2 nd band is “primer dimer” primers overlapping and amplifying themselves about 50bp or RNA that was not digested, near 121bp marker Other faint bands are when primers bind other places, “non-specific” amplification products