Lab 4abij DNA study
Purpose
The purpose of this lab was to learn how to make solutions. To find how DNA moves through substances. Also to learn how to find the amount of a chemical to add to make the correct concentration.
The purpose of this lab was to learn how to make solutions. To find how DNA moves through substances. Also to learn how to find the amount of a chemical to add to make the correct concentration.
Materials
Tris EDTA Sodium Chloride Analytical Balance Balance, tabletop milligram Ethanol 95% Loading Dye Electrophoresis Chamber |
Weigh Paper Graduated cylinder De- ionized water Salmon sperm DNA Beakers Micropipets and tips for p 20, 200, and 100 tubes 15 ml Ethidium Bromide Safety goggle, and gloves Power Source |
Glass Rods Pipet pumps 2 and 10 ml pipet pumps Lab Scoops Agarose Microwave Sharpie Hot hand protector Gel box TAE buffer Micro Centrifuge |
The molarity equation we used: (Molarity) x (Volume) x (Formula Weight). We used this equation to find how much of the solution we should add to get the right concentration.
Procedure
Making NaCl (Sodium Chloride)
1. Determine the mass of NaCl to make 5M. You will need 2.92 grams of NaCl
2. Place NaCl in 15 ml tube and add water to make 10 ml of it
3. Cap it. Label it. Store it.
Prepping TE buffer
1. Determine the amount of Tris and EDTA needed to make the right concentration You will need .158 grams of Tris and .037 grams of EDTA
2. Pour both solutions in a 250 ml beaker
3. add 80 ml of de-ionized water test pH of substance
4. put a couple drops of a strong base to bring the pH up to 8.0
5. Add more water till it gets to 100ml. Cap it. Label it. Store it.
Spooling the DNA
1. Fill the conical tube with 2 ml of TE
2. add 1ml of DNA and dilute with TE
3. Add 4ml of Ethanol to DNA slowly on the side to make a layer
4. The DNA will clump then spool it and put it in the tube with the 2ml of TE.
Making Agarose Gel
1. Make a 1xTAE solution by adding 12.5ml of 40x TAE to a graduated cylinder and filling it to 500ml
2. Mix 100ml of the TAE with .4 grams of agarose in a 250 ml glass flask, boil it in a microwave for 4 minutes on 50% pour
3. Let it cool and pour it in a taped off gel box, put the gel box combs in it, let it harden
Electrophoresis to study DNA
1. Take off tape and remove combs and put it in an electrophoresis chamber
2. Cover in TAE
3. Put 2 micro liter of the DNA and 4 microliters of loading die in small tube, run for 2 seconds in mini centrifuge
4. Use a micropipete to put solution in the well in the gel that the combs made, make sure that there are no air bubbles.
5. Plug gel in for 45 minutes, at 110 volts
6. Remove gel and place in new holding cell, stain with ethidium bromide overnight
7. Rinse off gel, observe under UV light
Making NaCl (Sodium Chloride)
1. Determine the mass of NaCl to make 5M. You will need 2.92 grams of NaCl
2. Place NaCl in 15 ml tube and add water to make 10 ml of it
3. Cap it. Label it. Store it.
Prepping TE buffer
1. Determine the amount of Tris and EDTA needed to make the right concentration You will need .158 grams of Tris and .037 grams of EDTA
2. Pour both solutions in a 250 ml beaker
3. add 80 ml of de-ionized water test pH of substance
4. put a couple drops of a strong base to bring the pH up to 8.0
5. Add more water till it gets to 100ml. Cap it. Label it. Store it.
Spooling the DNA
1. Fill the conical tube with 2 ml of TE
2. add 1ml of DNA and dilute with TE
3. Add 4ml of Ethanol to DNA slowly on the side to make a layer
4. The DNA will clump then spool it and put it in the tube with the 2ml of TE.
Making Agarose Gel
1. Make a 1xTAE solution by adding 12.5ml of 40x TAE to a graduated cylinder and filling it to 500ml
2. Mix 100ml of the TAE with .4 grams of agarose in a 250 ml glass flask, boil it in a microwave for 4 minutes on 50% pour
3. Let it cool and pour it in a taped off gel box, put the gel box combs in it, let it harden
Electrophoresis to study DNA
1. Take off tape and remove combs and put it in an electrophoresis chamber
2. Cover in TAE
3. Put 2 micro liter of the DNA and 4 microliters of loading die in small tube, run for 2 seconds in mini centrifuge
4. Use a micropipete to put solution in the well in the gel that the combs made, make sure that there are no air bubbles.
5. Plug gel in for 45 minutes, at 110 volts
6. Remove gel and place in new holding cell, stain with ethidium bromide overnight
7. Rinse off gel, observe under UV light
Data Analysis
Our final product did not actually work. We had many ideas why this did not work but the most likely reason it that the project didn't work was that one of the reagents was or went bad. We thought the stain went bad and it did because our teacher stored the stain we used last year thinking it would last forever, but it turned out it did go bad. That just shows you that one little mess up can ruin a project. After everything happened we ran a new gel with newly made athedium bromide and that proved to be what was wrong with it. Pic of our gel
Our final product did not actually work. We had many ideas why this did not work but the most likely reason it that the project didn't work was that one of the reagents was or went bad. We thought the stain went bad and it did because our teacher stored the stain we used last year thinking it would last forever, but it turned out it did go bad. That just shows you that one little mess up can ruin a project. After everything happened we ran a new gel with newly made athedium bromide and that proved to be what was wrong with it. Pic of our gel
Reflection
This project was fun because it was a four in a lab, but it was dis-heartening to find out that is didn't work in the end. This lab taught me so much like how to find the amount needed to make the right concentration. Also I learned how to use the analytic balance and how to make solutions. One pit was that it never actually worked. One peak was that my partner and a work so well together we finished an hour before everyone else. Overall I had a great together and it was interesting to learn everything.
This project was fun because it was a four in a lab, but it was dis-heartening to find out that is didn't work in the end. This lab taught me so much like how to find the amount needed to make the right concentration. Also I learned how to use the analytic balance and how to make solutions. One pit was that it never actually worked. One peak was that my partner and a work so well together we finished an hour before everyone else. Overall I had a great together and it was interesting to learn everything.