Purpose
Our purpose was to make 10 milliliters (mL) of 5 NaCl solution, and to make 100mL of TE buffer from 10 mM TRIS, 1 mM EDTA. Also we had to find out if DNA could be spooled out of solution, to find out what DNA looks like and its many unique properties. Finally we needed to find out what yield of DNA can be recovered during the isolation, to prepare and pour an agarose gel for DNA fragment analysis, to find out what the appearance of different DNA samples on an agarose gel.
Materials
Lab 4A:
analytical balance
tabletop milligram balance
weigh paper
weigh boat
lab scoops
sodium chloride
15 ml tubes
tube racks for 15 ml tubes
TRIS
EDTA
125 ml bottle
100 ml graduated cylinder
pH paper, wide/narrow-range
Hydrochloric acid
Sodium hydroxide
glass rods
Lab 4B:
50 ml beakers
salmon sperm DNA
2 - 20 ml pipet
pipet pump
micropipet (P-1000)
micropipet tips for P-1000
ethanol (95%)
glass rods
15 ml conical tubes (capped)
tube racks fro 15 ml conical tubes
permanent lab markers
Lab 4I:
TAE buffer concentrate (40x stock)
600 ml beaker
agarose
tabletop milligram balance
weigh paper
lab scoops
250 ml media bottle
permanent lab markers
microwave oven
hot hands protector
horizontal gel box for agarose gels
50 ml beakers
Lab 4J
horizontal Gel box for agarose gels
prepared agarose gel
TAE buffer concentrate (40x stock)
tube rack for 15 ml conical tubes
permanent lab markers
DNA samples
gel loading dye (6x)
micropipet (P-2-20)
micropipet (P-20-200)
micropipet tips for P-2-20
micropipet tips for P-20-200
microcentrifuge
power supply
ethidium bromide (0.5 micrograms/ml)
gloves
analytical balance
tabletop milligram balance
weigh paper
weigh boat
lab scoops
sodium chloride
15 ml tubes
tube racks for 15 ml tubes
TRIS
EDTA
125 ml bottle
100 ml graduated cylinder
pH paper, wide/narrow-range
Hydrochloric acid
Sodium hydroxide
glass rods
Lab 4B:
50 ml beakers
salmon sperm DNA
2 - 20 ml pipet
pipet pump
micropipet (P-1000)
micropipet tips for P-1000
ethanol (95%)
glass rods
15 ml conical tubes (capped)
tube racks fro 15 ml conical tubes
permanent lab markers
Lab 4I:
TAE buffer concentrate (40x stock)
600 ml beaker
agarose
tabletop milligram balance
weigh paper
lab scoops
250 ml media bottle
permanent lab markers
microwave oven
hot hands protector
horizontal gel box for agarose gels
50 ml beakers
Lab 4J
horizontal Gel box for agarose gels
prepared agarose gel
TAE buffer concentrate (40x stock)
tube rack for 15 ml conical tubes
permanent lab markers
DNA samples
gel loading dye (6x)
micropipet (P-2-20)
micropipet (P-20-200)
micropipet tips for P-2-20
micropipet tips for P-20-200
microcentrifuge
power supply
ethidium bromide (0.5 micrograms/ml)
gloves
Procedure
1st Day:
1. Determine mass of NaCl to be measured to give a 5 M concentration for 10 ml of the solution.
2. Put NaCl in 15 ml conical tube. Add distilled water until solution's volume is 10 ml.
3. Cap 15 ml conical tube. Label and keep in 4 degrees Celsius until ready to use.
4. Determine mass of TRIS to be measured to get correct concentration and volume in TE buffer.
5. Determine mass of EDTA to be measured to get correct concentration and volume in TE buffer.
6. Add TRIS and EDTA in correct amounts to 250-ml.
7. Add 80 ml of deionized water and mix until chemicals dissolved. Add NaOH to raise pH until in range (7.5 and 8.5). Use pH paper to determine pH. If solution too basic, add small volumes of 1 M of HCl to lower pH.
8. Add deionized water until total volume is 100 ml.
9. Pour buffer in 125-ml bottle, cap it, label it, and store at 4 degrees Celsius until ready to use.
2nd Day:
1. Mix 1 ml of DNA with 1 ml of TE buffer.
2. Dilute DNA with TE in beaker; observe.
3. Add NaCl (500 microliters of 5M NaCl).
4. Add 4 ml of Ethanol (trickle down side so careful not to mix solutions); observe.
5. Spool DNA.
6. Put DNA into new tube with 2 ml fresh TE and label.
3rd Day:
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.
4th Day:
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.
Data ANALYSIS
Unfortunately, our gels somehow did not have the DNA we thought they should have in them. We do not know how or why, but do to to an unknown reason the gels of our entire class and the other STEM class were lacking the DNA. My best guess at why they were lacking the DNA is some kind of error or miscommunication in the instructions. I think that the part where we most likely went wrong would be mixing the DNA with the dyes and not resuspending them properly or completely. Although it is unlikely that all sixty of us didn't properly complete that specific step, it is the most likely one I can think of. While brainstorming in class we thought that the DNA might have diffused out of the gels. This is unlikely because of the quantity of DNA is too high making diffusion almost impossible. The most likely explanation of why we were missing the DNA is that our reagents went bad. Regardless of why, our experiment is inconclusive.
reflection
I really liked this lab because in it we actually really learned a lot about how scientists use what they can to gather information. I liked working with my group because we worked quickly and efficiently and we all were good at both working together and splitting up and doing our own work to contribute. Our group had trouble in that sometimes we had members that were slightly controlling over who did what. Overall I think our group did great and i liked this project.