Lab 4a, 4b, 4l, & 4j (DNA Lab)
purpose:
1. Make 10 ml 5M NaCl solution
2. Make 100 ml TE buffer: 10 mM TRIS, 1 mM EDTA
3. To observe the appearance and unique properties of DNA by spooling it out of a solution.
4. To observe DNA or any part of DNA recovered during isolation.
5. Prepare and pour agarose gel for DNA fragment analysis.
6. Observe appearances of different DNA samples on an agarose gel.
2. Make 100 ml TE buffer: 10 mM TRIS, 1 mM EDTA
3. To observe the appearance and unique properties of DNA by spooling it out of a solution.
4. To observe DNA or any part of DNA recovered during isolation.
5. Prepare and pour agarose gel for DNA fragment analysis.
6. Observe appearances of different DNA samples on an agarose gel.
materials:
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procedure:
Lab 4a:
Part I
1. To make 10ml of 5M NaCl you do the molarity calculations to find that you use 2.92 g.
Molarity calculation formula: (Molarity) x (Volume) x (Formula Weight) = grams of substance needed
2. Measure out 2.92 g of NaCl, on a piece of weigh paper or boat, on the table top balance. Pour the NaCl into a test tube.
3. QS (top off) the tube with distilled water until it reaches 10ml, dissolve solution, and label/cap the tube.
Part II
1. To make the 100ml TE solution you have to mix Tris and EDTA. First do the molarity calculations for 10mM Tris - 0.1576 g, and 1mM EDTA - 0.0372 g. Then measure each one on weigh boats/paper in the analytic balance and add them to a beaker of 80 ml distilled water.
2. Test the pH, and add HCL to change the solution's pH until it's between 7.5 and 8.5. QS the solution with distilled water until it reaches 100ml.
3. Transfer the solution into a bottle, label, and place in a freezer until ready to use again.
Lab 4b:
1. Place 1 ml of DNA and 1 ml of the TE solution into a beaker [Calculated using (Concentration1)(Volume1) = (Concentration2)(Volume2), to find amount of DNA and TE].
2. Observe solution, viewed watery and clear, and add 500 microliters of 5 M NaCl. Then add 4 ml of ETOH to the solution by dripping it down the side of the tube, to create separate layers. Observe again, viewed opaque and slightly layered.
3. Spool the DNA out of the solution by twisting a glass rod in the solution. Move the spooled DNA into a new tube with 2 ml of TE, label/cap tube, and store in a freezer.
Lab 4i:
1. To create a 0.8% agarose gel, first calculate 1x TAE from 40x stock in 500ml by using (C1)(V1) = (C2)(V2) to find that 12.5 ml are needed. QS the solution with distilled water to 500ml.
2. Use the TAE information to find the amount of 0.8% agarose needed in 50 ml of 1x TAE. After multiplying the decimal of 0.8% agarose and 50 ml, its found that 0.4 g of agarose is needed. Use the analytical balance to measure the 0.4 g agarose on a weigh boat and mix into 50 ml of 1x TAE.
3. Heat solution in a microwave oven until all the agarose is dissolved. While heating and mixing the solution, tape each open side of the gel box to the same height as the other walls and place the gel box comb in the slits.
4. When the gel solution has cooled pour it into the gel box. Wrap the box in Saran Wrap and label.
Lab 4j:
Part I
1. Remove the tape from the box. Place the gel box in the gel tank and submerge it with the 1x TAE solution and carefully take out the comb.
2. Next place 2 ml of the DNA and 4 ml of 6x loading dye in a 1.7 ml tube. To mix the liquids and put them in one spot, place the tube, with a counter weight tube, in the mini centrifuge and have it spin for 2 seconds.
3. Use a micropipet to transfer the loading dye/DNA mixture into the wells left by the comb. Make sure there are no air bubbles when doing this and that you don't puncture the gel or it won't work.
Part II
1. Place the cover on the gel tank and plug it in the power source. Leave it plugged in for 45 minutes at 110 v.
2. Unplug the tank and take out the gel box. carefully transfer the gel into a new box and stain the gel with ethidium bromide for several hours. Wear protection such as gloves when handling the Ethidium Bromide because it contains carcinogens.
3. Rinse off the gel with distilled water and observe under a UV light box.
Part I
1. To make 10ml of 5M NaCl you do the molarity calculations to find that you use 2.92 g.
Molarity calculation formula: (Molarity) x (Volume) x (Formula Weight) = grams of substance needed
2. Measure out 2.92 g of NaCl, on a piece of weigh paper or boat, on the table top balance. Pour the NaCl into a test tube.
3. QS (top off) the tube with distilled water until it reaches 10ml, dissolve solution, and label/cap the tube.
Part II
1. To make the 100ml TE solution you have to mix Tris and EDTA. First do the molarity calculations for 10mM Tris - 0.1576 g, and 1mM EDTA - 0.0372 g. Then measure each one on weigh boats/paper in the analytic balance and add them to a beaker of 80 ml distilled water.
2. Test the pH, and add HCL to change the solution's pH until it's between 7.5 and 8.5. QS the solution with distilled water until it reaches 100ml.
3. Transfer the solution into a bottle, label, and place in a freezer until ready to use again.
Lab 4b:
1. Place 1 ml of DNA and 1 ml of the TE solution into a beaker [Calculated using (Concentration1)(Volume1) = (Concentration2)(Volume2), to find amount of DNA and TE].
2. Observe solution, viewed watery and clear, and add 500 microliters of 5 M NaCl. Then add 4 ml of ETOH to the solution by dripping it down the side of the tube, to create separate layers. Observe again, viewed opaque and slightly layered.
3. Spool the DNA out of the solution by twisting a glass rod in the solution. Move the spooled DNA into a new tube with 2 ml of TE, label/cap tube, and store in a freezer.
Lab 4i:
1. To create a 0.8% agarose gel, first calculate 1x TAE from 40x stock in 500ml by using (C1)(V1) = (C2)(V2) to find that 12.5 ml are needed. QS the solution with distilled water to 500ml.
2. Use the TAE information to find the amount of 0.8% agarose needed in 50 ml of 1x TAE. After multiplying the decimal of 0.8% agarose and 50 ml, its found that 0.4 g of agarose is needed. Use the analytical balance to measure the 0.4 g agarose on a weigh boat and mix into 50 ml of 1x TAE.
3. Heat solution in a microwave oven until all the agarose is dissolved. While heating and mixing the solution, tape each open side of the gel box to the same height as the other walls and place the gel box comb in the slits.
4. When the gel solution has cooled pour it into the gel box. Wrap the box in Saran Wrap and label.
Lab 4j:
Part I
1. Remove the tape from the box. Place the gel box in the gel tank and submerge it with the 1x TAE solution and carefully take out the comb.
2. Next place 2 ml of the DNA and 4 ml of 6x loading dye in a 1.7 ml tube. To mix the liquids and put them in one spot, place the tube, with a counter weight tube, in the mini centrifuge and have it spin for 2 seconds.
3. Use a micropipet to transfer the loading dye/DNA mixture into the wells left by the comb. Make sure there are no air bubbles when doing this and that you don't puncture the gel or it won't work.
Part II
1. Place the cover on the gel tank and plug it in the power source. Leave it plugged in for 45 minutes at 110 v.
2. Unplug the tank and take out the gel box. carefully transfer the gel into a new box and stain the gel with ethidium bromide for several hours. Wear protection such as gloves when handling the Ethidium Bromide because it contains carcinogens.
3. Rinse off the gel with distilled water and observe under a UV light box.
data results:
Our data results were invalid and could not be used, unfortunately.
data analysis:
For some reason, the DNA in the gels did not portray clearly the desired results (or any valid results at all). The unusual part of this was that this happened to everyone in our class. As a class, we brainstormed the possible reasons why all our experiments failed. Here were the possible causes:
1. Stained overnight and the DNA diffused out.
2. Denatured DNA.
3. Dye not loaded correctly.
4. Dye not resuspended before loading in gel.
5. Buffers not made correctly.
6. One of the reagents was bad.
After considering the possibilities, we decided that the most likely cause of the failure was a bad reagent. Dr. LB remade the ethidium bromide and ran the gels again, and it worked.
1. Stained overnight and the DNA diffused out.
2. Denatured DNA.
3. Dye not loaded correctly.
4. Dye not resuspended before loading in gel.
5. Buffers not made correctly.
6. One of the reagents was bad.
After considering the possibilities, we decided that the most likely cause of the failure was a bad reagent. Dr. LB remade the ethidium bromide and ran the gels again, and it worked.
reflection:
This lab had a few setbacks, such as the reagent problem, but I think it ran smoothly and efficiently. Some members in the group stepped forward into a leadership role at some points in the lab, and stepped back to let others take control at other times. Each member helped each other out and gave advice and input when the other was confused. In this way, we worked together nicely and the lab turned out well. We might have been able to improve upon our focus a little, but overall we accomplished a lot.