lab 4a, b, i, j - dna 8 / 15 / 14
Purpose
4a: Making Solutions for DNA Isolation
To make 10 mililiters (mL) of 5 M NaCl solution.
To make 100 mL of TE buffer: 10 mM TRIS, 1 mM EDTA (DNA storage solution)
4b: Pulling DNA out of Solution: DNA Spooling
Can DNA be spooled out of solution?
What does DNA look like?
What are some of its many unique properties?
What yield of DNA can be recovered during the isolation?
4i: Making Agarose Gels for Seperating and Analyzing DNA Fragments
To prepare and pour an agarose gel for DNA fragment analysis
4j: Using Gel Electrophoresis to Study DNA Molecules
What is the apperance of different DNA samples on an agarose gel?
To make 10 mililiters (mL) of 5 M NaCl solution.
To make 100 mL of TE buffer: 10 mM TRIS, 1 mM EDTA (DNA storage solution)
4b: Pulling DNA out of Solution: DNA Spooling
Can DNA be spooled out of solution?
What does DNA look like?
What are some of its many unique properties?
What yield of DNA can be recovered during the isolation?
4i: Making Agarose Gels for Seperating and Analyzing DNA Fragments
To prepare and pour an agarose gel for DNA fragment analysis
4j: Using Gel Electrophoresis to Study DNA Molecules
What is the apperance of different DNA samples on an agarose gel?
Materials
Lab 4a: Making Solutions for DNA Isolation
balance, analytical
balance, tabletop milligram
weigh paper, 7.6X7.6 cm
weigh boat 3.5"X3.5"
lab scoops
sodium chloride
tubes 15 mL, cappped
tube racks for 15 mL tubes
TRIS
EDTA, disodium salt
bottle, 125 mL
graduated cylinder, 100 mL
pH paper, wide/narrow-range
Hydrochloric acid
Sodium hydroxide
glass rods
Lab 4b: Pulling DNA out of solution: DNA spooling
beakers, 50 mL
DNA, salmon testes
pipet, 2 mL
pipet pump, blue
micropipet, P-1000
mircopipet tips for P-1000
Ethanol, 95%
glass rods
tubes, 15 mL capped
tube racks for 15 mL tubes
permanent lab marker pens
plastic beaker, 1L tripour
Lab 4i: Making agarose gels for separating and analyzing DNA fragments
TAE buffer concentrate, 40X
beakers, 600 mL
agarose
balance, tabletop milligram
weigh boat, 3.5"X3.5"
lab scoops
media bottle, 250 mL
permenent lab marker
glaases, saftey
gel box, horizontal, for agarose gels
beakers, 50 mL
Lab 4j: Using Gel Electrophoresis to Study DNA Moleculesgel box, horizantal, for aragose gels
tube rack for 1.7mL tubes
DNA samples (from previous labs)
micropipet, P-10
prepared aragose gels (from Lab 4h)
reaction tubes,1.7 mL
yeast DNA
micropipet, P-100
TAE buffer concentrate, 40X
permanent lab marker pens
gel loading dye, 10X
micro[pipet tips for p-10
balance, analytical
balance, tabletop milligram
weigh paper, 7.6X7.6 cm
weigh boat 3.5"X3.5"
lab scoops
sodium chloride
tubes 15 mL, cappped
tube racks for 15 mL tubes
TRIS
EDTA, disodium salt
bottle, 125 mL
graduated cylinder, 100 mL
pH paper, wide/narrow-range
Hydrochloric acid
Sodium hydroxide
glass rods
Lab 4b: Pulling DNA out of solution: DNA spooling
beakers, 50 mL
DNA, salmon testes
pipet, 2 mL
pipet pump, blue
micropipet, P-1000
mircopipet tips for P-1000
Ethanol, 95%
glass rods
tubes, 15 mL capped
tube racks for 15 mL tubes
permanent lab marker pens
plastic beaker, 1L tripour
Lab 4i: Making agarose gels for separating and analyzing DNA fragments
TAE buffer concentrate, 40X
beakers, 600 mL
agarose
balance, tabletop milligram
weigh boat, 3.5"X3.5"
lab scoops
media bottle, 250 mL
permenent lab marker
glaases, saftey
gel box, horizontal, for agarose gels
beakers, 50 mL
Lab 4j: Using Gel Electrophoresis to Study DNA Moleculesgel box, horizantal, for aragose gels
tube rack for 1.7mL tubes
DNA samples (from previous labs)
micropipet, P-10
prepared aragose gels (from Lab 4h)
reaction tubes,1.7 mL
yeast DNA
micropipet, P-100
TAE buffer concentrate, 40X
permanent lab marker pens
gel loading dye, 10X
micro[pipet tips for p-10
procedure
Lab 4a
Part I: Preparing 5M of NaCL
1. Use the molarity formula to figure that you must add 2.92 grams of NaCl to a conical tube
2. Add 10 ml of water to the conical tube.
3. Vertex then label. Keep for later use.
Part II: Preparing TE Buffer
1. Add 0.1576 grams of TRIS and 0.037224 grams of EDTA to beaker.
2. Add 80 ml of water to the same mixture.
3. Add HCI to solution until the pH level is inbetween 7.5 and 8.5.
4. Add more water till the solution is at 100 ml.
5. Label and keep for later use.
Lab 4b
Part I:
1. Put 1 mL of Salmon Sperm DNA and 1 mL of TE into a beaker
2. Add 5M of NaCl to the same beaker
3. Add 4 mL of ETOH to beaker. Do not mix.
4. Using a glass stir rod spool out the DNA
5. Place the spooled DNA into a conical tube and add 2 mL of TE
Lab 4i
Part I:
1. Add 0.4g agarose powder and QS TAE to 100 mL
2. Heat solution until the agarose has dissolved
3. Let solution cool and then pour it into the gel box. Then put the combs into the gel.
4. Let it set
Lab 4j
Part I:
1. Cover the gel in TAE
2. Add 20 uL of DNA and 4 mL of 6x loading dye into a tube
3. Centrifuge the solution
4. Using a micropipet, load the solution into the gel
5. Then apply electric charge to gel for approximately 45 minutes
6. Stain the gel with Ethidium Bromide
7. Rinse off and watch what happens
Part I: Preparing 5M of NaCL
1. Use the molarity formula to figure that you must add 2.92 grams of NaCl to a conical tube
2. Add 10 ml of water to the conical tube.
3. Vertex then label. Keep for later use.
Part II: Preparing TE Buffer
1. Add 0.1576 grams of TRIS and 0.037224 grams of EDTA to beaker.
2. Add 80 ml of water to the same mixture.
3. Add HCI to solution until the pH level is inbetween 7.5 and 8.5.
4. Add more water till the solution is at 100 ml.
5. Label and keep for later use.
Lab 4b
Part I:
1. Put 1 mL of Salmon Sperm DNA and 1 mL of TE into a beaker
2. Add 5M of NaCl to the same beaker
3. Add 4 mL of ETOH to beaker. Do not mix.
4. Using a glass stir rod spool out the DNA
5. Place the spooled DNA into a conical tube and add 2 mL of TE
Lab 4i
Part I:
1. Add 0.4g agarose powder and QS TAE to 100 mL
2. Heat solution until the agarose has dissolved
3. Let solution cool and then pour it into the gel box. Then put the combs into the gel.
4. Let it set
Lab 4j
Part I:
1. Cover the gel in TAE
2. Add 20 uL of DNA and 4 mL of 6x loading dye into a tube
3. Centrifuge the solution
4. Using a micropipet, load the solution into the gel
5. Then apply electric charge to gel for approximately 45 minutes
6. Stain the gel with Ethidium Bromide
7. Rinse off and watch what happens
data results
We completed the procedure and came to school the next day excited to observe the DNA in the gels and what had happened. When we came to school Dr. LB found that none of the DNA appeared. This was hard because at first it felt like the whole lab was for nothing, but we began to brainstorm what happened to try to fix it. After our brainstorm we came up with an idea to make a new stain and try again.
data analysis
As a whole, our class came up with some ideas.
Ideas of why the DNA didn't appear:
1. The overnight straining caused DNA to diffuse out with dye
2. Denatured DNA
3. We did not load DNA in to gel properly.
4. The stain had gone bad.
5. The dye was not resuspended before it was loaded into the gel
The likelihood each possibility:
1. Not likely because DNA is too small
2. Not likely because that is the purpose of the DNA
3. Not likely because that would mean every single did the lab wrong
4. Most likely because the chemicals in the stain could of deteriorated and gone bad
5. Not likely because that would mean every single did the lab wrong
Our solution was to create a new stain and try the experiment again. These were the results:
Ideas of why the DNA didn't appear:
1. The overnight straining caused DNA to diffuse out with dye
2. Denatured DNA
3. We did not load DNA in to gel properly.
4. The stain had gone bad.
5. The dye was not resuspended before it was loaded into the gel
The likelihood each possibility:
1. Not likely because DNA is too small
2. Not likely because that is the purpose of the DNA
3. Not likely because that would mean every single did the lab wrong
4. Most likely because the chemicals in the stain could of deteriorated and gone bad
5. Not likely because that would mean every single did the lab wrong
Our solution was to create a new stain and try the experiment again. These were the results:
REflection
At first the stain didn't work Dr. LB made a new stain and it worked.
My group worked well together, even though we we sometimes behind other groups and a little confused. I liked that we were able to see the DNA and everyone was excited to see it. One thing we struggled on was understanding why we were doing each step. I got most of them, but there were a few I didn't get. Sometimes we didn't make progress in a period and thats we were behind the other groups. We had a few flaws but overall I think we did good and we got the lab done completed and on time.
My group worked well together, even though we we sometimes behind other groups and a little confused. I liked that we were able to see the DNA and everyone was excited to see it. One thing we struggled on was understanding why we were doing each step. I got most of them, but there were a few I didn't get. Sometimes we didn't make progress in a period and thats we were behind the other groups. We had a few flaws but overall I think we did good and we got the lab done completed and on time.