Purpose:
a. To make 10 ml of 5M NaCl solution and to make 100 ml of TE buffer (10mM TRIS and 1mM EDTA)
b. To find out if DNA can be spooled, its appearance, its properties, and what yield of DNA can be recovered during isolation.
i. To create and prepare an agarose gel for DNA fragment analysis.
j. What appearance differences do separate DNA samples have on the agarose gel.
a. To make 10 ml of 5M NaCl solution and to make 100 ml of TE buffer (10mM TRIS and 1mM EDTA)
b. To find out if DNA can be spooled, its appearance, its properties, and what yield of DNA can be recovered during isolation.
i. To create and prepare an agarose gel for DNA fragment analysis.
j. What appearance differences do separate DNA samples have on the agarose gel.
Materials:
Analytical Balance Tabletop Balance Weigh Paper/Boat Scoops Sodium Chloride Capped Tubes(15ml) Tube Rack TRIS EDTA |
Sodium Hydrochloric Acid Sodium Hydroxide Granulated Cylinder(100ml) pH strips 50ml Beakers DNA(Salmon Testes) 2ml Pipet and Pump Micropipet, P-1000, and Tips 95% Ethanol |
Glass Rods TAE buffer, 40X 600ml Beakers Agarose 250ml bottle Microwave Oven Hot Hands Protector Safety Glasses Saran Wrap |
Gel Box and Gel Box Comb Gel Tank Power Supply Gloves Ethidium Bromide Microcentrifuge 6x Loading Dye Distilled Water |
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 analytical 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.
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 analytical 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 Analysis:
Unfortunately our lab didn't work because the DNA didn't appear when we looked for it under the UV light. As a class we brainstormed on reason why it may not have worked like if the DNA diffused out, we didn't load the DNA correctly, or one of the reagents went bad. We figured that the most logical reason it didn't work was that maybe the stain had gone bad. To test this theory we made a batch of the stain with the old 20,000x stock and one with a new 20,000x stock. We then tested the stains, each on a gel. After a day of staining, rinsing, and waiting a few more days our Lab finally worked. The DNA appeared to move through the gel at the same amount for each test. In the end the lab worked and we had a great example of DNA.
Reflection:
Besides the fact that the lab didn't work, I think my group did a good job working together and staying focused. We finish everything on time and even a few things early. My group could have done better at getting use to limited tools quicker since for some things we had to just wait and couldn't continue. It also would have helped if we had checked our substances better from the start because we had to redo one of our loading dyes. However we measured everything else perfectly and stayed on track. Throughout the project I think we worked very well and did a good job completing the lab, except for the part when they didn't work. In the end it worked and we did do everything right except for having a usable stain.
Unfortunately our lab didn't work because the DNA didn't appear when we looked for it under the UV light. As a class we brainstormed on reason why it may not have worked like if the DNA diffused out, we didn't load the DNA correctly, or one of the reagents went bad. We figured that the most logical reason it didn't work was that maybe the stain had gone bad. To test this theory we made a batch of the stain with the old 20,000x stock and one with a new 20,000x stock. We then tested the stains, each on a gel. After a day of staining, rinsing, and waiting a few more days our Lab finally worked. The DNA appeared to move through the gel at the same amount for each test. In the end the lab worked and we had a great example of DNA.
Reflection:
Besides the fact that the lab didn't work, I think my group did a good job working together and staying focused. We finish everything on time and even a few things early. My group could have done better at getting use to limited tools quicker since for some things we had to just wait and couldn't continue. It also would have helped if we had checked our substances better from the start because we had to redo one of our loading dyes. However we measured everything else perfectly and stayed on track. Throughout the project I think we worked very well and did a good job completing the lab, except for the part when they didn't work. In the end it worked and we did do everything right except for having a usable stain.