Dna LAbs: 4a, 4b, 4i, 4j
Purpose:
4A: Create 10mm of NaCl along with 100mL of TE buffer. Make the TE buffer using 10mM TRIS and 1mM EDTA.
4B: Find out what DNA's properties, can it be spooled, and what is the amount that can be recovered through isolation.
4I: Create and pour an agarose gel and prepare it for DNA Fragment Analysis.
4J: Describe the apperance of the DNA Samples in agarose gel.
Materials List:
- Tabletop Balance (mm)
- Analytical Balance
- Weigh Paper
- Weigh Boat
- Lab Scoops
- NaCl (Sodium Chloride)
- 15 mL Test Tubes
- TRIS
- EDTA
- 100 mL Graduated Cylinder
- 125 mL Bottle
- pH Test Strips
- Glass Stir Rods
- Sodium Hydroxide
- 2 mL Pipet
- 50 mL Beaker
- Salmon Sperm
- P-1000 Micropipet
- 95% Ethanol
- 40x TAE Buffer Concentrate
- 600 mL Beakers
- Agarose
- 250 mL Media Bottle
- Microwave
- Gel Box (Horizontal)
- Water Bath (65* Celcius)
- 1.7 mL Reaction Tubes
- Microcentrifuge
- Ethidium Bromide
- Power Source
Procedure:
Lab 4A:
1) Find mass of NaCl required for 5 M NaCl Solution.
2) Place the amount in a 15 mL Test Tube. Add water until the 10 mL mark.
3) Label the tube and place away for later.
4) Find amount of TRIS and EDTA for a TE buffer.
5) Add the reagent into a 250 mL beaker.
6) Add 80 mL of de-ionized water to the beaker.
7) Add Sodium Hydroxide to alter pH
8) QS to 100 mL
9) Label and place away for later.
Lab 4B:
1) Dilute DNA with TE within a beaker, Calculate with: C^1 V^1 = C^2 V^2. Record Observation.
(C = Concentration, V = Volume)
2) Add 500 microliters (ul) of 5 M NaCl.
3) Gently add 4 ml of ETOH to the solution.
4) Spool DNA with the Glass Stir Rod
5) Label and place away for later.
Lab 4I:
1) Find how much agarose is required to take TAE
2) Add agarose to 100 mL 1x TAE
3) Heat mixture until boiling. Let it cool.
4) Pour solution into the sealed gel box. Place comb within the box.
5) Label and place away for later.
Lab 4J:
1) Remove the comb and tape from the box.
2) Place the box in a gel tank and submerge in the TAE
3) Place 2 ul of DNA + 4 ul of 6x loading dye into 1.7 ml tube. place in mini centrifuge for 2 seconds.
4) Use a micropipet to move the mixture into wells within the gel created by th e comb. Check for air bubbles.
5) Plug in the gel tank to the power source at 100 v for 45 mins.
6) Remove the gel and transfer into new box. Stain with Ethidium Bromide overnight.
7) Rinse off and observe under UV light.
Lab Analysis:
When preforming these labs, everything went well until we stained the gel with the Ethidium Bromide. The stain was supposed to reveal the DNA within the gel but didn't. Fortunately, we weren't the only group to encounter this problem. Every other group had the same problem in their gels. We couldn't pin-point the exact cause of the problem and we ended up with the cinclusion that the problem lie with the stain that was used. It was the only logical reason we could find. Plus it was the same stain that was used last year and could have possibly been contaminated since then. We recovered from the problem by creating a new batch of Ethidium Bromide which allowed us to finish the lab.
Conclusion:
By isolating and purifying DNA like we did, it allows for endless amounts of manipulations to that DNA. It can manipulate it identify a person for anything including crimes or ancestry. This process just shows how complicated the human body is and how far technology has come as well.
Reflection:
Our group decided to split the work and place an even amount of work on everyone so that we could get done quickly and efficiently and hopefully also accident-free. With the work divided, the labs went by quick and easy until the last lab where my partner, yazeed, failed to create a proper stain. He had put air bubbles his portion of the stain and didn't realize it until he place the solution into the gel. We then had to extract the solution and create a new one. This set us back a little bit but it wasn't all that bad. It made me realize that mistakes do happen, and that you can't let them weigh you done.
4A: Create 10mm of NaCl along with 100mL of TE buffer. Make the TE buffer using 10mM TRIS and 1mM EDTA.
4B: Find out what DNA's properties, can it be spooled, and what is the amount that can be recovered through isolation.
4I: Create and pour an agarose gel and prepare it for DNA Fragment Analysis.
4J: Describe the apperance of the DNA Samples in agarose gel.
Materials List:
- Tabletop Balance (mm)
- Analytical Balance
- Weigh Paper
- Weigh Boat
- Lab Scoops
- NaCl (Sodium Chloride)
- 15 mL Test Tubes
- TRIS
- EDTA
- 100 mL Graduated Cylinder
- 125 mL Bottle
- pH Test Strips
- Glass Stir Rods
- Sodium Hydroxide
- 2 mL Pipet
- 50 mL Beaker
- Salmon Sperm
- P-1000 Micropipet
- 95% Ethanol
- 40x TAE Buffer Concentrate
- 600 mL Beakers
- Agarose
- 250 mL Media Bottle
- Microwave
- Gel Box (Horizontal)
- Water Bath (65* Celcius)
- 1.7 mL Reaction Tubes
- Microcentrifuge
- Ethidium Bromide
- Power Source
Procedure:
Lab 4A:
1) Find mass of NaCl required for 5 M NaCl Solution.
2) Place the amount in a 15 mL Test Tube. Add water until the 10 mL mark.
3) Label the tube and place away for later.
4) Find amount of TRIS and EDTA for a TE buffer.
5) Add the reagent into a 250 mL beaker.
6) Add 80 mL of de-ionized water to the beaker.
7) Add Sodium Hydroxide to alter pH
8) QS to 100 mL
9) Label and place away for later.
Lab 4B:
1) Dilute DNA with TE within a beaker, Calculate with: C^1 V^1 = C^2 V^2. Record Observation.
(C = Concentration, V = Volume)
2) Add 500 microliters (ul) of 5 M NaCl.
3) Gently add 4 ml of ETOH to the solution.
4) Spool DNA with the Glass Stir Rod
5) Label and place away for later.
Lab 4I:
1) Find how much agarose is required to take TAE
2) Add agarose to 100 mL 1x TAE
3) Heat mixture until boiling. Let it cool.
4) Pour solution into the sealed gel box. Place comb within the box.
5) Label and place away for later.
Lab 4J:
1) Remove the comb and tape from the box.
2) Place the box in a gel tank and submerge in the TAE
3) Place 2 ul of DNA + 4 ul of 6x loading dye into 1.7 ml tube. place in mini centrifuge for 2 seconds.
4) Use a micropipet to move the mixture into wells within the gel created by th e comb. Check for air bubbles.
5) Plug in the gel tank to the power source at 100 v for 45 mins.
6) Remove the gel and transfer into new box. Stain with Ethidium Bromide overnight.
7) Rinse off and observe under UV light.
Lab Analysis:
When preforming these labs, everything went well until we stained the gel with the Ethidium Bromide. The stain was supposed to reveal the DNA within the gel but didn't. Fortunately, we weren't the only group to encounter this problem. Every other group had the same problem in their gels. We couldn't pin-point the exact cause of the problem and we ended up with the cinclusion that the problem lie with the stain that was used. It was the only logical reason we could find. Plus it was the same stain that was used last year and could have possibly been contaminated since then. We recovered from the problem by creating a new batch of Ethidium Bromide which allowed us to finish the lab.
Conclusion:
By isolating and purifying DNA like we did, it allows for endless amounts of manipulations to that DNA. It can manipulate it identify a person for anything including crimes or ancestry. This process just shows how complicated the human body is and how far technology has come as well.
Reflection:
Our group decided to split the work and place an even amount of work on everyone so that we could get done quickly and efficiently and hopefully also accident-free. With the work divided, the labs went by quick and easy until the last lab where my partner, yazeed, failed to create a proper stain. He had put air bubbles his portion of the stain and didn't realize it until he place the solution into the gel. We then had to extract the solution and create a new one. This set us back a little bit but it wasn't all that bad. It made me realize that mistakes do happen, and that you can't let them weigh you done.