Question

1. Describe the functions of the following reagents in extraction of DNA from corn meal: proteinase K; guanidine HCI; SDS 2. Why is the ratio of the OD at 260 and 280 nm used to estimate DNA purity? 3. In one paragraph, summarize basic principles of PCR technique in your own words. List all the reagents you will need to perform a PCR experiment. Does this method tell you what genetic modifications were made? If yes, describe how you can tell the genetic modifications made. If no, describe what information you actually get in this lab. 4. Lab goals Test common food products for the presence of markers of genetic modifications Introduce and practice methods for DNA extraction, DNA quantitation, PCR and horizontal electrophoresis Background Two of the biggest challenges facing farmers are controlling weeds and diseases while maintaining crop yield and quality. It is important that these objectives are met cheaply, as the average acre yields 128 bushels of corn and the average price of corn has been $2.31/bushel over the last ten years1. That $295/acre needs to pay for the cost of seed, equipment, fertilizer, weed control, and the labor of the farmer (salary for the year!). Considering an acre 4,840 square yards, the most practical method for achieving good weed and disease control, crop yield and quality are mass-methods, not hoeing by hand. This has historically meant the widespread use of pesticides and herbicides to control unwanted weeds and insects. A new approach to these issues is through the production of genetically modified plants. There are two common types of genetically modified crops: insect resistant (Bt) and herbicide resistant (Round-Up Ready) "Bt" is short for Bacillus thuringiensis, a soil bacterium whose spores contain a protein (Cry), which is broken down in an insect's gut to release a toxin, known as a delta-endotoxin. This toxin binds to and creates pores in the intestinal lining, resulting in an ion imbalance, paralysis and after a few days, death of the insect.2 Several different Cry genes, also known as "Bt genes" have been identified. They are effective against different orders of insects, or affect the insect gut in slightly different ways. The genes which have been approved for use in human food products denature under high heat and are easily digested by gastric enzymes. Recently, Taco Bell brand taco shells were removed from the market for containing an unapproved Cry protein, Cry9C. This protein is heat stable and resistant to degradation by trypsin, two characteristics of potential food allergens.3 Herbicide resistant varieties contain a bacterial enzyme which degrades the herbicide so that it is not toxic to the plant To make a genetically modified plant, several components are needed in addition to the gene for the protein of interest. First, a method for getting the gene into the plant is needed. Most commonly a plasmid from Agrobacterium tumefaciens called Ti is used. Once the gene is in the plant, it must be correctly transcribed and translated. This requires a promoter sequence and species-specific codon usage. Usually a powerful promoter from Cauliflower Mosaic Virus (CaMV 35 S) is used. In this lab, we will be isolating DNA from several corn meal products possibly containing an herbicide resistance gene using an affinity purification column from New England Biolabs (NEB) Monarch PCR &DNA Cleanup Kit. Then we will test the extracted DNA for the presence of the CaMV 35S promoter using PCR·This will indicate that the source material either was genetically modified OR that the plant was infected by CaMV (it's unlikely that corn would be infected bya cauliflower virus on its own.) While this will indicate whether the product contains transgenic plant material, t does not tell what the trans gene (i.e. the gene that was inserted) is PCR stands for "Polymerase Chain Reaction". This procedure was originally developed by Kerry Mullis, who won a Nobel Prize for this work. A heat resistant polymerase from a thermophilic bacterium, Thermus aquaticus, (Taq), is used to replicate the DNA located between two primers. Two short oligonucleotide primers are annealed to denatured (single strand) DNA by using hybridization conditions which ensure that only perfectly (or nearly so) complementary primers will anneal to the DNA sequence. The primers are then extended using DNA polymerase and the four deoxynucleotide triphosphates (dNTPs), generating two duplex DNA copies of the targeted region. The DNA strands are separated by heating the sample, but the Tag polymerase is not denatured. These three steps (annealing, elongation, and thermal denaturation of the DNA) constitute one "cycle" of PCR DNA replication. Multiple cycles are performed, with each copy of the desired sequence being copied each cycle. Thus, after 40 cycles of amplification, the target sequence is amplified (ideally) over 1 billion-fold Safety notes Ethidium bromide is a potent mutagen and should be handled with care. Gloves, goggles, and lab coat Gels containing ethidium bromide should be disposed appropriately. UV eye protection must be used with transluminators that do not include built in safety shields. worn whenever work ing with solutions or gels containing ethidium bromide. Pre-lab questions Read through the procedure and related contents in the textbook, answer the following questions be fore the lab period. Failure to submit your pre-lab assignment will prevent you from doing the experiment I. Describe the functions of the following reagents in extraction of DNA from com meal: proteinase K; guanidine HCl: SDS 2. Why is the ratio of the OD at 260 and 280 nm used to estimate DNA purity? 3. In one paragraph, summarize basic principles of PCR technique in your own words. List all the reagents you will need to perform a PCR experiment. Does this method tell you what genetic modifications were made? If yes, describe how you can tell the genetic modifications made. If no, describe what information you actually get in this lab 4. Procedure Week 1 1) Isolate DNA from four source materials For corn meal samples: mix 0.3 g ofcom meal with 600 μL ofddH20 in a 2 mL microfuge tube. Add 860 μし0fextraction buffer ( 10 mM Tris, pH 7.5, 150 mM NaCL 2 mM EDTA. 1% SDS), 100 μ L of 5 M guanidine HCI, and 40 uL of 20 mgmL proteinase K. BE VERY CAREFUL TO USE THE CORRECT PIPPETS AND TIPS, AND DO NOT CONTAMINATE THE SAMPLES!!! a) K. c FOR ALL SAMPLES: Incubate at 55-60 C for 30 min with intermittent shaking. During this time, work with your discussion group on the GMO reading set. d) Allow samples to cool to room temperature for 5 minutes. e Centrifuge at 13,000 rpm for 10 minutes in a tabletop microcentrifuge. DNA cleanup (adapted from NEB #T1030) a) 2) Obtain a DNA Cleanup column and a collection tube from the instructor. All centrifugation steps are carried out at 13,000 rpm b) For each sample, take 100 μ1 of the cleared supermatant, and dilute with 200 μ1 of DNA c) Insert column into collection tube and load sample onto column. Spin for 1 minute, then d) Re-insert column into collection tube. Add 200 μ1 DNA Wash Buffer and spin for 1 minute. e Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the f) g) The column and be discarded, and keep the flow-through in the microfuge tube for the Quantitation of amount and purity of DNA obtained from purification Cleanup Binding Buffer discard flow-through. Discard flow-through. Repeat this step one more ti me column does not come into contact with the flow-through. If in doubt, re-spin for 1 minute. Add 20 μ1 of DNA Elution Buffer to the center ofthe matrix. Wait for 1 minute, then spin for 1 minute to elute DNA next step 3) Mix 1 μ1 of purified DNA sample with 199 ㎕ Qubit in a clear 500-μ1 Qubit assay tube Votex for 2-3 sec. Incubate at room temperature for 2-3 min. a) b) Calibrate the instrument with two standards (performed by the instructor) c) Insert the tube with your sample into the instrument. Press "Run Samples". On the assay screen, select the sample volume of 1 ul. d) Press "Read tube". When reading is completed (3 sec), remove sample tube. e The instrument displays the results on the assay screen. The top value (in large font) is the concentration of the original sample bottom value is the dilution concentration. le. This is the concentration you should record. The

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