Lab 4a, 4b, 4i, & 4j:
Purpose:
To learn the technique of spooling DNA as well as making solutions.
hypothesis:
If correct procedure is performed then our solutions and DNA gels should have successful results.
Overall materials:
- Analytical Balance
- Tabletop Milligram Balance - 7.6 x 7.6 cm Weigh Paper - 3.5 x 3.5 Weigh Boat - Lab Scoops - Sodium Chloride -15 mL Capped Tubes - Tube Racks - TRIS - EDTA - Disodium Salt - 125 mL Bottle - 100 mL Graduated Cylinder - Glasses |
.- pH Paper
- Hydrochloric Acid - Sodium Hydroxide - Glass Rods - 50 mL Beakers - Salmon Sperm DNA - 2 mL Pipette - P-1000 Micropipette, and tips - 95% Ethanol - Permanent Marker Pens - 1L Tripour Plastic Peaker - 40X TAE Buffer Concentrate - 600 mL Beakers - Agarose |
- 250 mL Media Bottle
- Microwave Oven - Hot Hands Protector - Horizontal Gel Box - 65 degree Celcius water bath - Reaction Tubes, 1.7 mL - DNA Samples - Loading Dye: 6x concentration - Micro Pipettes - Microcentrifuge - Power Supply - Ethidium Bromide - Gel Photo Imaging System - Gloves |
Procedure:
Lab4 a & b Procedure:
1. Calculate molarity:
~~Given: 1 mole = 6.02 x 10^23 ; 5 moles = 5 (6.02 x 10^23 particles) / 1 Litre ; (Molarity) (Volume) (Formula weight) = g substance needed~~
NaCl: 2.92 grams
TRIS: 0.158 grams
EDTA: 0.037 grams
2. Create TE Solution by combining TRIS and EDTA.
3. Dilute DNA with TE solution.
4. Add NaCl.
5. Add alcohol by trickling it down the side of the flask.
6. Spool DNA
7. Put the spooled DNA into a different tube and add 2 mL of TE solution.
Lab4i Procedure:
1. Prepare 100 mL solution of 0.8% agarose in 1X TAE buffer solution.
2. Weigh out the required mass of powdered agarose in a weigh boat. Add it to a 250-mL media bottle.
3. Measure out enough TAE buffer to prepare a total of 100 mL of agarose and buffer mixed together. Swirl to mix.
4. Cap the media bottle and swirl the flask to suspend the agarose in the buffer.
5. To dissolve the agarose, microwave for 4 minutes at 50% power. Wait for the solution to boil.
6. Place the hot dissolved agarose solution on a fireproof lab tabletop and let cool to 65 degrees Celsius before pouring into a gel tray.
7. Place a six-well comb into the notches at the end of the gel tray. This will create the necessary wells for the next experiment.
8. Place the gel (on the gel tray) into a gel box.
9. Pour 1X TAE buffer into the gel box and completely submerge the gel.
10. Gently pull the comb out of the gel and make sure the wells are not broken or cracked.
Lab 4j: Using Gel Electrophoresis to Study DNA MoleculesProcedure:
1. Prepare the gel and gel box for loading.
2. Carefully secure gates of the gel tray. Make sure the 1X TAe buffer covers the gel by at least 1 centimeter.
3. Obtain 1.7-mL tubes.
4. Add 20 micro liters of salmon sperm DNA and add 3 micro liters of 6X DNA loading dye. Spin the sample in a minicentrifuge.
5. Load the salmon sperm DNA sample into the wells using a micro pipette.
6. Connect the electrodes of the gel box to the power supply and run the gel at 110 Volts for 45 minutes.
7. Run until you can see the front loading dye halfway down the gel.
8. Cover the gel with EtBr (ethinium bromide) and stain the gel for 20 minutes. Then pour off the EtBr, cover with deionized water, and observe the gel on a UV light box.
9. Analyze the contents of each well, size of the standards, the samples, and the DNA bands.
1. Calculate molarity:
~~Given: 1 mole = 6.02 x 10^23 ; 5 moles = 5 (6.02 x 10^23 particles) / 1 Litre ; (Molarity) (Volume) (Formula weight) = g substance needed~~
NaCl: 2.92 grams
TRIS: 0.158 grams
EDTA: 0.037 grams
2. Create TE Solution by combining TRIS and EDTA.
3. Dilute DNA with TE solution.
4. Add NaCl.
5. Add alcohol by trickling it down the side of the flask.
6. Spool DNA
7. Put the spooled DNA into a different tube and add 2 mL of TE solution.
Lab4i Procedure:
1. Prepare 100 mL solution of 0.8% agarose in 1X TAE buffer solution.
2. Weigh out the required mass of powdered agarose in a weigh boat. Add it to a 250-mL media bottle.
3. Measure out enough TAE buffer to prepare a total of 100 mL of agarose and buffer mixed together. Swirl to mix.
4. Cap the media bottle and swirl the flask to suspend the agarose in the buffer.
5. To dissolve the agarose, microwave for 4 minutes at 50% power. Wait for the solution to boil.
6. Place the hot dissolved agarose solution on a fireproof lab tabletop and let cool to 65 degrees Celsius before pouring into a gel tray.
7. Place a six-well comb into the notches at the end of the gel tray. This will create the necessary wells for the next experiment.
8. Place the gel (on the gel tray) into a gel box.
9. Pour 1X TAE buffer into the gel box and completely submerge the gel.
10. Gently pull the comb out of the gel and make sure the wells are not broken or cracked.
Lab 4j: Using Gel Electrophoresis to Study DNA MoleculesProcedure:
1. Prepare the gel and gel box for loading.
2. Carefully secure gates of the gel tray. Make sure the 1X TAe buffer covers the gel by at least 1 centimeter.
3. Obtain 1.7-mL tubes.
4. Add 20 micro liters of salmon sperm DNA and add 3 micro liters of 6X DNA loading dye. Spin the sample in a minicentrifuge.
5. Load the salmon sperm DNA sample into the wells using a micro pipette.
6. Connect the electrodes of the gel box to the power supply and run the gel at 110 Volts for 45 minutes.
7. Run until you can see the front loading dye halfway down the gel.
8. Cover the gel with EtBr (ethinium bromide) and stain the gel for 20 minutes. Then pour off the EtBr, cover with deionized water, and observe the gel on a UV light box.
9. Analyze the contents of each well, size of the standards, the samples, and the DNA bands.
data analysis:
How did our dna band appear?
Due to our stain going bad we did not have a DNA band to observe. We are working to salvage the experiment and continue on with a new stain.
Possible Errors in our Experiment:
-We stained our DNA overnight and the DNA could have diffused out
-This is unlikely because we used fairly big DNA
-Stain went bad after sitting for too long
- This is more likely because the stain was not made freshly
Possible Errors in our Experiment:
-We stained our DNA overnight and the DNA could have diffused out
-This is unlikely because we used fairly big DNA
-Stain went bad after sitting for too long
- This is more likely because the stain was not made freshly
After making a new stain and continuing on with the experiment we had a successful result as shown in the picture at the top of the page.
conclusion:
what is the value of isolating dna from a solution and separating it in a gel?
- Research and lab purposes
Being able to isolate DNA can be very beneficial for testing and manipulating to find out more about how DNA works to then apply it to human DNA and figure why certain traits and diseases occur.
Being able to isolate DNA can be very beneficial for testing and manipulating to find out more about how DNA works to then apply it to human DNA and figure why certain traits and diseases occur.
how could these techniques be used in a biotech or forensics lab?
We could use these techniques in a lab to manipulate the DNA of' certain organisms. We could figure out how DNA works in different organisms as well.
reflection:
My group and I worked very well together there was no arguments or tension and we all shared the responsibilities of the lab.
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Overall my group seemed to pipette rather well. One member had a little trouble to start out with but we helped guide her and she got it after a few tries. My group made solutions rather well too. We just listen to the instructions given and use common sense which works out well.
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We could have been more efficient by double checking the directions which would have prevented us from miscalculating the amount of DNA to put.
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