Lab 4
Materials:
Hypothesis:
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Procedure:
part I: preperation of 5 M of NaCl
1. determine the mass of NaCl to be measured. In notebook draw and explain a diagram of the solution.
2. place NaCl in a 15mL capped, conical tube. Cap it. Slowly add H2O, while stirring, until a final volume of the 10mL is reached.
3. pour the mixture into a 15mL capped, conical tube. Cap it. Label it with the sample name, concentration, date, and technicians initials. Store at 4 celcius until ready to use.
part II: preperation of TE buffer
1. determine the mass of TRIS to be measured to give the correct concentration and volume in the final TE buffer. Show final calculations in your notebook.
2. determine the mass of EDTA to be measured to give correct concentration and volume in the final TE buffer. Record in notebook.
3. measure out the TRIS and EDTA, and add them to a 250mL beaker.
dna spooling
1. using the TE buffer as the solvent and the C1 V1 = C2 V2 equation, determine how to make 2mL of 2mg/mL from a 4mg/mL salmon sperm DNA solution. Record in notebook.
2. prepare the diluted salmon sperm DNA solution in a prechilled, clean 50mL beaker. You will be using this 2mg/mL DNA solution in the next step.
3. describe the appearance, color, vicoscity, etc, of the 2mg/mL slamon sperm DNA. Add these data to the data table.
4. using a micro pipet, add 500uL of 5 M NaCl solution. Mix by swirling.
5. keep everything as cold as possible. Slowly trickle 4mL of ETOH down the side of the beaker containing the DNA and NaCl. Do not mix the alcohol and DNA layers.
6. observe the interface between the two solutions. You should see a layer of alcohol on the top of the layer containing the DNA and NaCl. Do not mix the two layers. Describe what the layers look like. Add these data to the data table.
7. place the glass rod at the interface of the two layered solution. During spooling, you will force ETOH from the top layer down into the bottom DNA layer and pull the DNA out as it mobes away from the ETOH.
8. holding the beaker tilted toe the right 45degrees, wind the DNA that comes out of the solution onto the rod. these are not single DNA molecules, but thousands of molecules. watch the interface as you rotate the rod.
9. examine and touch the DNA on the rod. Record the appearance of DNA, including color, texture, and other characteristics. Add these data to the data table. Touching the samples exposes it to DNase enzyme, which chops DNA. Record.
10. shake the ethanol of the spooled DNA. Touch it to a paper towel. Get rid of as much ethanol as you can without losing the DNA sample.
11. scrape the spooled DNA into 2mL of TE buffer in a labeled, capped, conical, 15mL tube. Record
12. store at 4degrees c for at least one week. During that time, the DNA should go back into the solution. Record. After that it should be ready to use for indicator testing or gel electrophoresis.
procedure-
1. Determine the mass of agarose to be measured to put in the 1X TAE (TRIS-acetate -EDTA).
2. Add agarose to 100 ml 1X TAE in erlenmeyer flask.
3. Heat to boil and dissolve (heat-swirl-heat-swirl until clear) in a microwave oven.
4. Let cool until you can touch the erlenmeyer flask for a few seconds.
5. Prepare the gel mold by taping the opposite open ends and putting the two combs in.
6. Pour 1X TAE and agarose solution into gel mold and let cool.
procedure-
1. Remove tape from gel. Place in gel tank.
2. Pour TAE over gel until covered. Gently remove combs.
3. Prepare samples with micropipet (P-2-20):
- 20 ml DNA and 4 microliters 6x loading dye. Spin 2 seconds in centrifuge.
4. Load samples onto gel with micropipet (P-20-200).
5. Put cover on gel tank and plug into power supply.
6. Run at 110V for approximately 45 minutes.
7. Stain for several hours with Ethidium Bromide
8. Rinse and observe with light.
Conclusion
part I: preperation of 5 M of NaCl
1. determine the mass of NaCl to be measured. In notebook draw and explain a diagram of the solution.
2. place NaCl in a 15mL capped, conical tube. Cap it. Slowly add H2O, while stirring, until a final volume of the 10mL is reached.
3. pour the mixture into a 15mL capped, conical tube. Cap it. Label it with the sample name, concentration, date, and technicians initials. Store at 4 celcius until ready to use.
part II: preperation of TE buffer
1. determine the mass of TRIS to be measured to give the correct concentration and volume in the final TE buffer. Show final calculations in your notebook.
2. determine the mass of EDTA to be measured to give correct concentration and volume in the final TE buffer. Record in notebook.
3. measure out the TRIS and EDTA, and add them to a 250mL beaker.
dna spooling
1. using the TE buffer as the solvent and the C1 V1 = C2 V2 equation, determine how to make 2mL of 2mg/mL from a 4mg/mL salmon sperm DNA solution. Record in notebook.
2. prepare the diluted salmon sperm DNA solution in a prechilled, clean 50mL beaker. You will be using this 2mg/mL DNA solution in the next step.
3. describe the appearance, color, vicoscity, etc, of the 2mg/mL slamon sperm DNA. Add these data to the data table.
4. using a micro pipet, add 500uL of 5 M NaCl solution. Mix by swirling.
5. keep everything as cold as possible. Slowly trickle 4mL of ETOH down the side of the beaker containing the DNA and NaCl. Do not mix the alcohol and DNA layers.
6. observe the interface between the two solutions. You should see a layer of alcohol on the top of the layer containing the DNA and NaCl. Do not mix the two layers. Describe what the layers look like. Add these data to the data table.
7. place the glass rod at the interface of the two layered solution. During spooling, you will force ETOH from the top layer down into the bottom DNA layer and pull the DNA out as it mobes away from the ETOH.
8. holding the beaker tilted toe the right 45degrees, wind the DNA that comes out of the solution onto the rod. these are not single DNA molecules, but thousands of molecules. watch the interface as you rotate the rod.
9. examine and touch the DNA on the rod. Record the appearance of DNA, including color, texture, and other characteristics. Add these data to the data table. Touching the samples exposes it to DNase enzyme, which chops DNA. Record.
10. shake the ethanol of the spooled DNA. Touch it to a paper towel. Get rid of as much ethanol as you can without losing the DNA sample.
11. scrape the spooled DNA into 2mL of TE buffer in a labeled, capped, conical, 15mL tube. Record
12. store at 4degrees c for at least one week. During that time, the DNA should go back into the solution. Record. After that it should be ready to use for indicator testing or gel electrophoresis.
procedure-
1. Determine the mass of agarose to be measured to put in the 1X TAE (TRIS-acetate -EDTA).
2. Add agarose to 100 ml 1X TAE in erlenmeyer flask.
3. Heat to boil and dissolve (heat-swirl-heat-swirl until clear) in a microwave oven.
4. Let cool until you can touch the erlenmeyer flask for a few seconds.
5. Prepare the gel mold by taping the opposite open ends and putting the two combs in.
6. Pour 1X TAE and agarose solution into gel mold and let cool.
procedure-
1. Remove tape from gel. Place in gel tank.
2. Pour TAE over gel until covered. Gently remove combs.
3. Prepare samples with micropipet (P-2-20):
- 20 ml DNA and 4 microliters 6x loading dye. Spin 2 seconds in centrifuge.
4. Load samples onto gel with micropipet (P-20-200).
5. Put cover on gel tank and plug into power supply.
6. Run at 110V for approximately 45 minutes.
7. Stain for several hours with Ethidium Bromide
8. Rinse and observe with light.
Conclusion