Lab 4 a, b, i, and j - 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.
Materials
Lab 4a
analytical balance
tabletop milligram balance
weigh paper
weigh boat
lab scoops
sodium chloride
15 ml conical tubes
tube racks for 15 ml conical tubes
TRIS
EDTA (disodium salt)
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
Lab 4a
Part I: Preparing 5M of NaCL
1. Measure out 2.92 g of NaCl and add to tube
2. QS water to (add water until you reach) 10 mL
3. Label for later use
Part II: Preparing TE Buffer
1. Add 0.1576 g of TRIS and 0.037224 g of EDTA to a beaker
2. Add 80 mL of deionized water to solution
3. Add HCl to raise pH or NaOH to lower pH until solution has a pH between 7.5 and 8.5
4. QS water to 100 mL
5. Label for later use
Lab 4b
1. Add 1 mL DNA and 1 mL TE into a beaker.
2. Add 5M NaCl
3. Add 4 mL ETOH by pouring down side (forms two layers)
4. Using a glass rod, spool DNA
5. Place DNA in a tube with 2 mL TE
Lab 4i
1. Add 0.4g agarose powder and QS TAE to 100 mL
2. Heat solution until agarose is dissolved.
3. Let solution cool and then pour into gel box (with combs)
Lab 4j
1. Submerge gel and electrodes in TAE
2. Add 20 uL DNA and 4 mL of 6x loading dye into a tube
3. Centrifuge solution
4. Load solution into gel
5. Apply charge to gel for approximately 45 minutes
6. Stain with Ethidium Bromide, rinse, and observe
Data Reflection
After completing the lab, Dr. LB used a detector to see the DNA and the DNA did not show up inside the gels. After this info we took some time in class to brainstorm what went wrong and this is what we found.
Data Analysis
As a class, we came up with a few reasons on why the DNA did not appear
1. Staining overnight caused the DNA to diffuse out.
2. Denatured DNA
3. Did not load the DNA properly into the gel.
4. The stain was bad.
5. Dye not re-suspended before loading in gel.
Likelihood of these possibilities happening
1. Unlikely because the DNA is too small to diffuse out.
2. Unlikely because it is the stain's purpose.
3. Unlikely because that would mean every single group did the lab wrong.
4. Most likely because overtime the chemicals in the stain could have gone bad.
5. Unlikely because that would mean every single group did the lab wrong.
Reflection
My group worked well together, even if we sometimes got lost or were a little behind other groups. I liked that we were all into the lab and wanted to see the DNA. Something that we could improve upon would be focus, for example, we got carried away and did not make any progress on the lab on that day or, sometimes, we would leave somebody behind. Overall, I think there was definitely room for improvement, but we did well enough to complete the lab successfully.
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.
Materials
Lab 4a
analytical balance
tabletop milligram balance
weigh paper
weigh boat
lab scoops
sodium chloride
15 ml conical tubes
tube racks for 15 ml conical tubes
TRIS
EDTA (disodium salt)
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
Lab 4a
Part I: Preparing 5M of NaCL
1. Measure out 2.92 g of NaCl and add to tube
2. QS water to (add water until you reach) 10 mL
3. Label for later use
Part II: Preparing TE Buffer
1. Add 0.1576 g of TRIS and 0.037224 g of EDTA to a beaker
2. Add 80 mL of deionized water to solution
3. Add HCl to raise pH or NaOH to lower pH until solution has a pH between 7.5 and 8.5
4. QS water to 100 mL
5. Label for later use
Lab 4b
1. Add 1 mL DNA and 1 mL TE into a beaker.
2. Add 5M NaCl
3. Add 4 mL ETOH by pouring down side (forms two layers)
4. Using a glass rod, spool DNA
5. Place DNA in a tube with 2 mL TE
Lab 4i
1. Add 0.4g agarose powder and QS TAE to 100 mL
2. Heat solution until agarose is dissolved.
3. Let solution cool and then pour into gel box (with combs)
Lab 4j
1. Submerge gel and electrodes in TAE
2. Add 20 uL DNA and 4 mL of 6x loading dye into a tube
3. Centrifuge solution
4. Load solution into gel
5. Apply charge to gel for approximately 45 minutes
6. Stain with Ethidium Bromide, rinse, and observe
Data Reflection
After completing the lab, Dr. LB used a detector to see the DNA and the DNA did not show up inside the gels. After this info we took some time in class to brainstorm what went wrong and this is what we found.
Data Analysis
As a class, we came up with a few reasons on why the DNA did not appear
1. Staining overnight caused the DNA to diffuse out.
2. Denatured DNA
3. Did not load the DNA properly into the gel.
4. The stain was bad.
5. Dye not re-suspended before loading in gel.
Likelihood of these possibilities happening
1. Unlikely because the DNA is too small to diffuse out.
2. Unlikely because it is the stain's purpose.
3. Unlikely because that would mean every single group did the lab wrong.
4. Most likely because overtime the chemicals in the stain could have gone bad.
5. Unlikely because that would mean every single group did the lab wrong.
Reflection
My group worked well together, even if we sometimes got lost or were a little behind other groups. I liked that we were all into the lab and wanted to see the DNA. Something that we could improve upon would be focus, for example, we got carried away and did not make any progress on the lab on that day or, sometimes, we would leave somebody behind. Overall, I think there was definitely room for improvement, but we did well enough to complete the lab successfully.