Student Guide - DNA I



Detecting Genetically Modified Food by PCR

Most Americans would probably be surprised to learn that more than 60% of fresh vegetables and processed foods sold in supermarkets today, have been altered by direct gene transfer. Over 150 million acres of farmland worldwide are used to grow genetically modified (GM) crops. The most widely planted GM crops are corn, soybeans, and cotton.

This laboratory uses a rapid method to isolate DNA from plant tissue and to genotype a soy plant using the polymerase chain reaction (PCR) method. The 35S promoter, which drives expression of the glyphosate resistance gene, is identified in soy plants that are resistant to the herbicide "Roundup”. Resistance correlates with an insertion allele that is readily separated from the wild-type allele by electrophoresis on an agarose mini-gel. The technique can then be extended to assay common foods for molecular evidence of genetic modification.

Objectives/Goals:

This laboratory provides practical insight into:

• the relationship between genes, proteins, and traits.

• three methods (DNA extraction, PCR, and gel electrophoresis) that are commonly used in biological research.

• gene transfer technology and its impact on society.

INTRODUCTION

Methods in genetic engineering are producing a revolution in agriculture. Genes that encode for herbicide resistance, insect resistance, draught tolerance, frost tolerance, and other traits have been added to numerous plants of commercial importance- including strawberries, soy, corn, potatoes, rice, wheat, and canola.

A common approach to transferring new genes into different plant species makes use of the pathogenic plant bacterium Agrobacterium tumefaciens. In the wild, infection by this bacterium causes plant tumors, called crown galls. These tumors provide nutrients for the bacteria’s successful colonization of the plant. Galls are produced by the expression of genes contained on Agrobacterium’s tumor inducing (Ti) plasmid. The Ti plasmid contains a region of oncogenic DNA, flanked by direct repeats, that is transferred to the host cell. This mobile region of the Ti plasmid, called T (transfer) DNA, inserts randomly into the plant cell genome. Once inserted into the plant genome, the infected cell begins to express genes located on the T-DNA. The host cell then expresses genes on the T DNA needed by the pathogen. By placing DNA of interest within a modified T DNA region, researchers are able to use Agrobacterium to introduce foreign DNA into plants.

Agrobacterium-mediated gene transfer involves the use of two plasmids and is referred to as a “binary system”. First, the gene of interest is cloned into a small plasmid. This plasmid contains the direct repeat regions of the Ti plasmid, which allows efficient transfer of DNA into the plant cell genome. A polylinker containing multiple recognition sites allows one to splice a gene of interest between the direct repeats. The gene is inserted adjacent to a strong promoter-such as the 35S promoter from cauliflower mosaic virus. The plasmid also contains a selectable marker, the kanamycin-resistance gene, which allows successfully transformed plants to be identified by antibiotic selection. After subcloning the gene of interest, the plasmid is purified from E. coli and inserted into a strain of Agrobacterium that contains the second plasmid of the binary system. This plasmid is a larger, modified form of Ti, which carries genes required for infection and T-DNA transfer.

The genetically modified Agrobacterium is then used to infect a host plant. In Arabidopsis, ovules can be transformed simply by dipping early stage flowers in a suspension of Agrobacterium. After fertilization, a small proportion of seeds will produce genetically modified offspring.

Agriculture has been greatly impacted by the advances in gene transfer methods. One example has been the introduction of a gene that provides resistance to the broad-spectrum herbicide glyphosate, commonly known as “Roundup”. Crops genetically altered to possess this gene are referred to as “Roundup Ready”. These crops are resistant to the herbicide, while the invasive weeds are not. The major advantages of the "Roundup Ready” system include better weed control, reduction of crop injury, higher yield, and lower environmental impact than traditional herbicide systems.

REFERENCES

Castle, L.A., Siehl, D.L., Gorton, R., Patten, P.A., Chen, Y.H., Bertain, S., Cho, H.J., Wong, N.D., Liu, D., Lassner, M.W. (2004). Discovery and Directed Evolution of a Glyphosate Tolerance Gene. Science 304: 1151-1154.

Edwards, K., Johnstone, C. and Thompson, C. (1991). A Simple and Rapid Method for the Preparation of Plant Genomic DNA for PCR Analysis. Nucleic Acids. Res.19: 1349.

Stalker, D.M., McBride, K.E., Maiyj, L.D. (1988). Herbicide Resistance in Transgenic Plants Expressing a Bacterial Detoxification Gene. Science 242: 419-423.

Vollenhofer, S., Burg, K., Schmidt, J., Kroath, H. (1999). Genetically Modified Organisms in Food Screening and Specific Detection by Polymerase Chain Reaction. J. Agric. Food Chem. 47: 5038-5043

REAGENTS, SUPPLIES, AND EQUIPMENT

|Reagents |Supplies and Equipment |

|Wild-type and Roundup Ready soybean seed |Seed growing tray |

|Assorted dry food products |Planting container |

| |Potting soil |

|Edward’s buffer, 4 mL | |

|Isopropanol, 1.5 mL |Pellet pestles |

|Tris/EDTA (TE) buffer, 400 μL |Permanent markers |

| |1.5 mL microcentrifuge tubes |

|35S1/35S2 primer/loading dye mix*, 75 μL |Micropipets and tips (for measuring volumes from 2.5 μL to 900 μL) |

|Tub5/Tub3 primer/loading dye mix*, 75 μL |Microcentrifuge tube racks (or empty pipet tip boxes) |

|Ready-To-GoTM PCR beads |Microcentrifuge |

|Mineral oil, 5 mL (depending on thermal cycler) |Vortex (optional) |

| | |

|Marker, pBR322/BstN1* (9.75 μg), 130μL |Ice buckets with crushed ice |

|10X TBE, 300 mL |Thermal cycler |

|Agarose, 2 g |Water bath or heating block for boiling samples |

|Ethidium bromide (1 μg/mL), 250 mL |Gel electrophoresis chamber and power supply |

|or |Staining trays |

|CarolinaBLU( gel and buffer stain, 7 mL |Latex gloves |

|CarolinaBLU( final stain, 250 mL |White light box (to visualize DNA with CarolinaBLU) |

| |UV transilluminator (to visualize DNA with ethidium bromide) |

|*Store in a -20(C freezer | |

LAB FLOW AND SUMMARY

|Note: You must plant the soy seed 2-3 weeks prior to performinging the lab. |

This lab can be broken into three parts:

I. Isolating DNA from soy plant tissue and dry food products using Edward's buffer.

II. Amplifying the 35S promoter and tubulin locus by PCR. During this step, specific primers are used to analyze the wild-type and the transformed plants having the 35S promoter.

III. Analyzing the amplified DNA by agarose gel electrophoresis.

The following table will help you to plan and integrate the three parts of the experiment.

|Part |Day |Time | Activity |

| | | | |

|Preparation of |2-3 weeks |15-30 min. |Plant soy seeds |

|soy plants |before lab | | |

| | | | |

| | | | |

|I. DNA Isolation | 1 |30 min. |Pre-lab: Set up student stations |

| | |30-60 min. |Isolate soy DNA |

|II. PCR Amplification | 2 |30-60 min. |Pre-lab: Set up student stations |

| | |15-30 min. |Set up PCR reactions |

| | |70+ min. |Post-lab: Amplify DNA in thermal cycler |

|III. Analyzing Amplified | 3 |30 min. |Prepare agarose gel solution and cast gels |

|DNA by Gel | | | |

|Electrophoresis | | | |

| | 4 |30 min |Load DNA samples into gels |

| | |30+ min. |Electrophorese samples |

| | |20+ min. |Post-lab: Stain gels |

| | |20 min. to |Post-lab: De-stain gels |

| | |overnight | |

| | |20 min. |Post-lab: Photograph gels |

| | | | |

| | | | |

| | | | |

| | | | |

| | | | |

GROWING SOY

The table below provides a list of items for growing soy plants. Most of the supplies are from Hummert but many of them can be substituted with products that are easily obtainable from a hardware store.

|Item |Description |Supplier |

|Shelving |Any shelves can be used that can house fluorescent light fixtures|Hardware store |

| |so that lights are 12-18 inches from the plants. | |

|Fluorescent light fixtures and bulbs |Light fixtures should hold at least two 40 watt fluorescent |Hardware store |

| |bulbs. The lights should be “daylight” lights as opposed to cool | |

| |white lights. | |

|Timer |A timer is needed if plants are to be maintained on a cycle of 16|Hardware store |

| |hours of light and 8 hours of dark. Plants can also be grown in | |

| |24 hours of light if a timer is not available. | |

|Soil |The potting soil to grow soy is Metro Mix 200 (Catalog # | |

| |10-0325). Fertilizer and insecticide can be added as required. | |

|Trays |Soy plants are watered from above so planting pots should be | |

| |placed in plastic tray with holes (Catalog # 11-3000-1). The | |

| |industry standard for trays is a 10” X 20” plastic flat. The | |

| |planting pot inserts fit directly into these trays. | |

|Pots |The inserts for the trays commonly used contain 8 planting pots | |

| |(Catalog # 11-0250-1). There are also other inserts that can be | |

| |used if a different number of planting pots are required. | |

Planting Seeds

Obtain wild-type and Roundup Ready soy seed. Plant seeds as described below and allow for a 2-week growth period.

1. Place the planting pots into a plastic tray containing holes. The holes will allow excess water to drain from the soil when it is initially dampened. Fill the planting pots evenly with soil but do not pack the soil tightly. Note: For the best results, obtain or make a potting soil formulated specifically for growing soy seed.

2. Plant the seeds 0.5 inches below the soil using your finger. Leave 3 to 4 inches of space between them to allow optimal growth and to easily visualize the plant phenotype.

3. Place the planting container into the growing tray. Water the plants from above to prevent the soil from drying out. However, do not allow the soil to remain soggy.

4. Grow the plants close to a sunny window at room temperature or slightly warmer.

5. Harvest plant tissue for PCR as soon as the plant’s first leaves become visible. This should be about 2 weeks after planting – depending on light and temperature conditions.

PART I: ISOLATING DNA FROM SOY AND DRY FOOD PRODUCTS

Pre-lab Notes

Dry Food Products

Dry food products work best using the DNA extraction protocol outlined below. Food products should contain either soy or corn as an ingredient. Products that have been tested successfully using this procedure include Doritos brand tortilla chips, Tostitos blue corn chips, Betty Crocker Bacos, Jiffy corn muffin mix, Pepperidge Farm Sausalito cookies, Almased multi-protein powder, and Meow Mix cat food.

Pre-lab Set Up

Each station serving two students working as a team should have:

Wild-type and Roundup Ready soy plants

Dry food product

Edward's buffer, 4 mL

Isopropanol, 1.5 mL

Tris/EDTA (TE) buffer, 400 μL

6- 1.5 mL microcentrifuge tubes

Permanent marker

3- Disposable pellet pestles

20-200 μL micropipet and tips

100-1,000 μL micropipet and tips

Microcentrifuge

Water bath or heating block to boil samples

Vortex (optional)

Ice bucket with crushed ice

Procedure

1. Obtain one wild-type and one Roundup Ready soy plant. From each plant, take two pieces of leaf tissue approximately 1/4 inch in diameter. (The large end of a 1,000 μL pipet tip will punch disks of this size.) Place the leaf tissue in separate microcentrifuge tubes, and label with plant type and your group number.

2. For dry food products, obtain a 2-3 mm piece of food product and place in a separate microcentrifuge tube, and label with sample type and group number.

3. Add 100μL of Edward’s buffer to each tube containing the plant or food material. Grind the plant tissue or food product forcefully in the microcentrifuge tubes using separate pellet pestles. Grind for approximately 1 minute. The plant tissue sample should become green when it is fully ground.

4. Add 900 μL of Edward's buffer to each tube containing the ground sample. Grind briefly to remove tissue from the pellet pestle and to liquify any remaining pieces of sample.

5. Vortex the tubes for 5 seconds, by hand or machine. Boil the samples for 5 minutes in a water bath or heating block. Note: Be sure to monitor the samples as the eppendorf tube lids may open as the tubes heat.

6. Microcentrifuge the tubes containing the ground plant tissue or food sample for 2 minutes. After 2 minutes any insoluble material should form a tight pellet at the bottom of the tubes.

7. Transfer 350 μL of each supernatant to a fresh tube. The supernatant contains the desired DNA; make sure not to disturb the pelleted material when transferring the supernatant. (This is best accomplished by pipetting several times using a medium micropipet set at 100 μL.)

8. Add 400 μL of isopropanol to each of the DNA-containing supernatants. Mix, and leave at room temperature for 3 minutes. This step precipitates the DNA.

9. Microcentrifuge the tubes with the isopropanol and supernatant for 5 minutes with the hinge of tubes pointing outward.

10. After centrifugation, the pellet should be located at the bottom-side of the tubes underneath the hinge. It may be teardrop shaped or appear as small, scattered granules. Don’t be concerned if you can’t see a pellet. Carefully pour off the supernatant, then completely remove the remaining liquid with a medium pipet set at 100 μL.

11. Air dry the pellets for 10 minutes to remove any remaining isopropanol.

12. After drying, resuspend each DNA pellet in 100 μL of TE buffer. Pipet in and out, taking care to wash down the side of the tube underneath the hinge, where the DNA has accumulated during centrifugation.

13. Centrifuge the DNA/TE solution for 1 minute to pellet any material that did not go into solution. You will use 2.5 μL of the supernatant as the template DNA for the PCR reactions.

14. DNA may be used immediately or stored at -20(C. Keep the DNA on ice during use.

PART II: AMPLIFYING DNA BY PCR

Pre-lab Notes

This procedure uses two PCR reactions to analyze each plant or food product. One primer set (35S1/35S2) amplifies the 35S promoter of cauliflower mosaic virus. This promoter is used to drive expression of the glyphosate resistance gene used in Roundup Ready crops. The second set (Tub5/Tub3) amplifies a fragment of a tubulin gene that is present in all plants. Therefore, the primer set amplifying the tubulin gene is useful as a positive control for template DNA quality. It helps insure that any lack of amplification by the first primer set is a result of the absence of the 35S promoter – not a problem of template quantity or quality. The PCR products from the two primer sets are similar in size: 187 bp for the Tub5/Tub3 primer set and 162 bp for the 35S1/35S2 primer set. Therefore, these PCR products must be loaded in separate lanes of a 2% agarose gel during the electrophoresis part of this experiment.

Ready-To-Go PCR BeadsTM

Each PCR bead contains reagents so that when brought to a final volume of 25 μL the reaction contains 2.5 units of Taq polymerase, 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2, and 200 (M of each dNTP.

Primer/Loading Dye Mix

This mix incorporates the appropriate primer pair (0.25 picomoles/μL of each primer), 13.9% sucrose, and 0.0082% cresol red. The inclusion of loading dye components, sucrose and cresol red, allows the amplified product to be directly loaded into an agarose gel for electrophoresis.

Setting Up PCR Reactions

The lyophilized Taq polymerase in the Ready-To-Go PCR Bead™ becomes active immediately upon addition of the primer/loading dye mix. In the absence of thermal cycling, “nonspecific priming” allows the polymerase to begin generating erroneous products, which can show up as extra bands in gel analysis. Therefore, work quickly. Be sure the thermal cycler is set and have all experimenters set up their PCR reactions as a coordinated effort. Add primer/loading dye mix to all reaction tubes, then add each student template, and begin thermal cycling immediately. Hold reactions on ice until ready to load into the thermal cycler.

Thermal Cycling

PCR amplification from crude cell extracts is biochemically demanding, and requires the precision of automated thermal cycling. However, amplification of the 35S and tubulin loci is not complicated by the presence of repeated units. Therefore, the recommended amplification times and temperatures will work adequately for all types of thermal cyclers.

To hand amplify, simply set up three constant temperature water baths (or heat blocks) at 94(C, 60(C, and 72(C. Secure the student reactions in a test tube rack, and rotate the rack successively through the three baths for 30 seconds each. Stop after 32 cycles.

Pre-lab Set up

Each station serving two students working as a team should have:

Wild-type and Roundup Ready soy DNA, from Part I (on ice)

Food Product DNA, from Part I (on ice)

75 μL 35S1/35S2 primer/loading dye mix (on ice)

75 μL Tub5/Tub3 primer/loading dye mix (on ice)

6 Ready-To-Go PCR Beads™ (in reaction tubes)

Permanent marker

1-20 μL micropipet and tips

20-200 μL micropipet and tips

Thermal cycler

Mineral oil, 5 mL (depending on thermal cycler)

Ice bucket with crushed ice

Procedure

1. Use a micropipet with a fresh tip to add 22.5 μL of the 35S1/35S2 primer/loading dye mix to each of 3 PCR tubes containing a Ready-To-Go PCR Bead™. Tap the tube very gently with your finger to dissolve the bead. Label each tube with primer set and group number. Then label one tube “WT” (wild-type), one tube “RR” (Roundup Ready.), and one tube “FP” (food product).

2. Use a micropipet with a fresh tip to add 22.5 μL of the Tub5/Tub3 primer/loading dye mix to each of 3 PCR tubes containing a Ready-To-Go PCR Bead™. Tap the tube very gently with your finger to dissolve the bead. Label each tube with primer set and group number. Then label one tube “WT” (wild-type), one tube “RR” (Roundup Ready.), and one tube “FP” (food product).

3. Use a micropipet with a fresh tip to add 2.5 μL of wild-type soy DNA (from Part I) to each of the two reaction tubes marked “WT”. Mix by gently pipeting up and down. If necessary, pool the reagents by pulsing in a microcentrifuge or by sharply tapping the tube bottom on the lab bench.

4. Use a micropipet with a fresh tip to add 2.5 μL of Roundup Ready soy DNA (from Part I) to each of the two reaction tubes marked “RR”. Mix by gently pipeting up and down. If necessary, pool the reagents by pulsing in a microcentrifuge or by sharply tapping the tube bottom on the lab bench.

5. Use a micropipet with a fresh tip to add 2.5 μL of food product DNA (from Part I) to each of the two reaction tubes marked “FP”. Mix by gently pipeting up and down. If necessary, pool the reagents by pulsing in a microcentrifuge or by sharply tapping the tube bottom on the lab bench.

6. If necessary, add one drop of mineral oil to the top of the reactants in the PCR tubes. Be careful not to touch the dropper tip to the tube or reactants, or subsequent reactions will be contaminated with DNA from your preparation. Note: Thermal cyclers with heated lids do not require the use of mineral oil.

7. Store all samples on ice or in the freezer until you are ready to amplify. Program the thermal cycler for 32 cycles with the following cycle profile. The program may be linked to a 4°C hold program after the 32 cycles are completed.

Denaturation step: 94(C 30 sec

Annealing step: 60(C 30 sec

Extension step: 72(C 30 sec

8. After cycling, store the amplified DNA at -20°C until you are ready to continue with Part III.

PART III: ANALYZING AMPLIFIED DNA BY GEL ELECTROPHORESIS

Pre-Lab Notes

An objective in this experiment is to let students determine the genotype of the individual plants. The students can also pool their data and use the segregation ratio of the genotypes to determine the genotype of the parental plant.

Cresol Red Loading Dye

The cresol red and sucrose in the primer mix function as loading dye, so that amplified samples can be loaded directly into an agarose gel. This is a nice time saver. However, since it has relatively little sugar and cresol red, this loading dye is more difficult to use than typical loading dyes. So, encourage students to load very carefully.

DNA Size Markers

Plasmid pBR322 digested with the restriction endonuclease BstN I is an inexpensive marker and produces fragments that are useful as size markers in this experiment. The size of the DNA fragments in the marker are 1,857 bp, 1,058 bp, 929 bp, 383 bp, and 121 bp. Use 20 μL of this DNA ladder per gel. Other markers or a 100 bp ladder may also be substituted.

Viewing and Photographing Gels

View and photograph gels as soon as possible after appropriate destaining. Over time, small-sized PCR products diffuse through the gel and become unclear. If kept refrigerated in a very small amount of distilled or deionized water, CarolinaBLU™ stained gels will retain their integrity for months.

Pre-lab Set Up

Depending upon your situation, you may wish to prepare the 1X TBE buffer, the 2% agarose, or the agarose gels ahead of time for your students. Instructions on how to prepare the 1X TBE buffer, the 2% agarose, and the agarose gels have been included with the student instructions should you decide to have your students perform these procedures. The set up below is described as though the instructor prepared the reagents, but not the gel, ahead of time.

Each station serving two students working as a team should have:

Wild-type soy 35S and tubulin PCR products from Part II (on ice)

Roundup Ready soy 35S and tubulin PCR products from Part II (on ice)

Food product 35S and tubulin PCR products from Part II (on ice)

pBR322/BstNI markers (on ice)

2% agarose in 1X TBE

1X TBE buffer

1 μg/mL ethidium bromide or

CarolinaBLU( staining solution

2-20 μL micropipet and tips

20-200 μL micropipet and tips

Electrophoresis chamber and power supply

Staining tray

Transilluminator with camera

Ice bucket with crushed ice

Procedure

1. Prepare a 1X concentration of TBE by diluting 300 mL of 10X concentrated stock into 2,700 mL of deionized or distilled water. Mix thoroughly.

2. Prepare a 2% agarose gel in 1X TBE by adding 2 g of agarose to 100 mL of 1X TBE in a 500 mL flask or beaker. Heat flask in a boiling water bath (approximately 15 minutes) or in a microwave oven (approximately 4 minutes) until the agarose is completely dissolved. You should no longer see agarose particles floating in solution when the beaker is swirled. Allow the agarose to cool to 55-65(C. You should be able to touch the container without burning yourself while pouring it into the gel tray.

3. Seal the ends of the gel tray with masking tape and insert the comb. Pour cooled agarose solution into the tray to form a gel approximately one quarter inch thick. Allow the gel to solidify completely. The gel should appear cloudy when completely solidified. This takes approximately 20 minutes.

4. Place the gel into the electrophoresis chamber, and add enough 1X TBE buffer to cover the surface of the gel. Carefully remove the comb, and add additional 1X TBE buffer to just cover and fill in wells, creating a smooth buffer surface. (Do not add more buffer than necessary. Too much buffer above the gel channels electrical current over the gel, increasing running time.)

5. Use a micropipet with a fresh tip to add 25 μL of each of the sample/loading dye mixtures into your assigned wells of a 2% agarose gel. (If you used mineral oil during PCR, pierce your pipet tip through the layer of mineral oil to withdraw the PCR products and leave the mineral oil behind in the original tube.) Important: Expel any air from the tip before loading, and be careful not to push the tip of the pipet through the bottom of the sample well.

6. Load 20 μL of the molecular weight marker (pBR322/BstN1) into one well.

7. Run the gels at 130 V for approximately 30 minutes. Adequate separation will have occurred when the cresol red dye front has moved at least 50 mm from the wells.

8. Once the loading dye has migrated an appropriate distance through the gel, process the gel by soaking it in stain. If you are using ethidium bromide, stain for 15 minutes. If you are using CarolinaBlu(, see below. Use gloves when handling ethidium bromide or anything that has ethidium bromide on it. Ethidium bromide is a known mutagen and care should be taken when using and disposing of it.

9. View gel using transillumination and photograph.

Staining with CarolinaBLU(

To stain gels following electrophoresis, cover the gel with the CarolinaBLU( Final stain and let sit for 20-30 minutes. Agitate gently (optional). Pour the stain back into the bottle to be used again. (The stain can be used 6-8 times.) Cover the gel with deionized or distilled water to destain. Use distilled or deionized water since the chloride ions present in tap water can partially remove the stain from the DNA bands and will cause the staining to fade. Change the water 3-4 times over the course of 30-40 minutes. Agitate the gel occasionally. Bands that are not immediately present will become more apparent with time and will reach their maximum visibility if the gel is left to stain overnight in just enough stain to cover the gel. Gels left overnight in a large volume of water may destain too much.

CarolinaBLU( can also be used to stain the DNA while the gel is being run. The staining will not be as intense as the final stain, and final staining will still be required. However, staining while the gel is running may slightly increase the sensitivity of the stain and may allow the students to visualize their results prior to the end of the gel run.

To stain the gel while it is running, add the CarolinaBLU( Gel and Buffer Stain in the amounts indicated below. Note that the amount of stain added is dependent upon the voltage used for electrophoresis. Do not use more stain than recommended. This may precipitate the DNA in the wells and create artifact bands. Gels containing CarolinaBLU( may be prepared one day ahead of the lab day if necessary. However, gels stored longer tend to fade and lose their ability to stain DNA bands during electrophoresis.

Use the table below for the addition of CarolinaBLU( gel and buffer stain to agarose solutions.

|Voltage |Agarose Volume |Stain volume |

|50 Volts |50 mL | 80 μL (2 drops) |

| |300 mL | 480 μL (12 drops) |

| |400 mL | 640 μL (16 drops) |

Use the table below for the addition of CarolinaBLU( stain to the 1X TBE buffer.

|Voltage |Buffer Volume |Stain volume |

|50 Volts |500 mL |960 μL (24 drops) |

| |2.6 liters | 5 mL (125 drops) |

RESULTS AND DISCUSSION

1. Observe the photograph of the stained gel containing your sample and those from other students. Orient the photograph with wells at the top. Interpret each lane of the gel. Use the sample gel pictured below to help you.

[pic]

a. Scan across the photograph of your gel and others as well to get an impression of what you see in each lane. You should notice that virtually all student lanes contain one or two prominent bands.

b. Now locate the lane containing the pBR322/BstN I markers on the left hand side of the gel. Working from the well, locate the bands corresponding to each restriction fragment: 1,857bp, 1,058 bp, 929 bp, 383 bp, and 121 bp (may be faint or not visible at all). Alternatively, locate the lane containing the 100 bp ladder on the right hand side of the gel. These DNA markers increase in size in 100 bp increments starting with the fastest migrating band of 100 bp.

c. The amplification product of the 35S promoter (162 bp), which has been amplified using the 35S1/35S2 primers, should align between the 121 bp marker and the 383 bp marker of pBR322/BstNI markers (or between the 100 bp and 200 bp markers of the 100 bp ladder). The amplification product of the tubulin gene (187 bp), which has been amplified using the Tub5/Tub3 primers, should align between the 121 bp marker and the 383 bp marker (or between the 100 bp and 200 bp markers of the 100 bp ladder).

d. Establish the exact size of your PCR products as follows.

i. Measure the distance that each marker DNA band migrated from the sample well by measuring from the front of each well to the front edge of each band. The sizes of the DNA fragments in your pBR322/BstNI marker are as follows: 1,857bp, 1,058 bp, 929 bp, 383 bp, and 121 bp. Should you use another marker, adjust the numbers accordingly.

ii. Set up semi-log graph paper with the x-axis as the distance migrated by the DNA fragments and the y-axis (the logarithmic axis) as base pair length of the fragments. Plot the distance migrated versus base-pair length for each marker DNA fragment and connect the data points with a line – this line will be referred to as the “standard curve.” There is a linear relationship between the distance that a DNA band travels and the log of its molecular weight. Since weight of a DNA fragment is proportional to the number of base pairs in the fragment, base pairs are frequently used in place of molecular weight when these types of calculations are done with DNA. Also note that since you are plotting the molecular weight (in this case base pairs) on the log scale it is not necessary to take the log of the molecular weight prior to graphing.

iii. Measure and record the distances migrated by the DNA bands in your PCR reaction. To determine the size of your DNA bands, first locate the position on the x-axis that indicates the distance migrated by that band. Then, use a ruler to draw a vertical line from this point to its intersection with the “standard curve.” Next, extend a horizontal line from this intersection point to the y-axis. The number on the y-axis is the calculated base-pair size of your PCR product.

e. It is common to see a second band lower on the gel. This diffuse (fuzzy) band is "primer dimer," an artifact of the PCR reaction that results from the primers overlapping one another and amplifying themselves. Primer dimer is approximately 50 bp, and should be in a position ahead of the 121 bp marker.

f. Additional faint bands, at other positions on the gel, occur when the primers bind to chromosomal loci other than tubulin or 35S and give rise to "nonspecific" amplification products.

2. How would you interpret a lane in which you observe primer dimer, but not the 164 bp or 162 bp bands?

The presence of primer dimer confirms that the reaction contained all the components necessary for amplification, but there was insufficient template to amplify target sequences.

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