DNA Labs: 4a, b,i,j
Purpose: 4a: To make 10 milliliters of 5 M NaCl solution, and to make 100 mL of TE buffer out of: 10 mM TRIS and 1 mM EDTA.
4b: What are DNA's properties (particularly it's appearance), can it be spooled, and how much can be recovered from isolation?
4i: To prepare and pour an agarose gel for DNA fragment analysis.
4j: What is the appearance of different DNA samples on an agarose gel?
Master list of materials:
Procedure:
Lab 4a:
Lab 4b:
Lab 4i:
Lab 4j:
Data Analysis:
In actuality, our experiment wasn't a success. We followed all of the steps up until #7, since our DNA somehow wasn't showing up after we stained them overnight. Instead, we had to come up with possible problems and solutions. Since everyone had the same result, we could rule out individual errors and doing the procedure wrong. It had to be something that was universal. The reagents maybe? They all seemed to react as they were supposed to, the stain was the only problem. Ah, then it could be the ethidium bromide. Our teacher had used the same stock from last year, assuming that it could be stored indefinitely. But, we don't know if being exposed to sunlight or something else could have corrupted it. So, we should make a new batch of ethidium bromide and test it.
Update: After restaining the gel, we were able to get tangible results, as shown in the picture below.
Conclusion:
The process of isolating and purifying DNA on a gel is very valuable to anyone who has to deal with genetics or DNA. DNA can only be read if it is purified, otherwise it is like reading a book with coffee spilled on it, it is impossible. Once a scientist has purified the DNA, they can manipulate it however they wish. Forensic scientists can use it to match with the sequences in their database, and find people who were at a crime and possibly a suspect. Anyone trying to make a hybrid life-form could use purified DNA. It is the building block of who we are. The uses for it are limitless.
Reflection:
Our group's philosophy was: the more, the less work for each. With six people, we got everything done really fast and without a large amount of effort from any one or two people. With one or two people doing small things, we were done with the current task with lots of time to spare, all six of us. If that's not efficient, I don't know what is. The only downside to this fact was that if one member of our group messed up, it hung up a lot of people. Since we were all supposed to make a DNA-Loading Dye mixture and then inject it into the gel, when one member of our group had to do the whole thing over we were delayed a whole period. Other than that though, it was a fun lab that was made even better by a good, efficient group. As for the delay, practice will make (near) perfect.
4b: What are DNA's properties (particularly it's appearance), can it be spooled, and how much can be recovered from isolation?
4i: To prepare and pour an agarose gel for DNA fragment analysis.
4j: What is the appearance of different DNA samples on an agarose gel?
Master list of materials:
- Analytical Balance
- Tabletop Balance (milligrams)
- 7.6 cm x 7.6 cm Weigh Paper
- 3.5" x 3.5" Weigh Boat
- Lab Scoops
- Sodium Chloride
- Capped 15 mL Tubes
- Tube Racks
- TRIS
- EDTA Disodium Salt
- 125 mL Bottle
- 100 mL Graduated Cylinder
- pH Test Strips
- Sodium Hydroxide
- Glass Rods
- 50 mL Beakers
- Salmon Sperm
- 2 mL Pipette
- P-1000 Micropipette, (tips included)
- 95% Ethanol
- 1L Tripour Plastic Peaker
- Sharpies
- 40X TAE Buffer Concentrate
- 600 mL Beakers
- Agarose
- 250 mL Media Bottle
- Microwave Oven
- Hot Hands Protector
- Horizontal Gel Box
- 65 degree Celcius Water Bath
- Reaction Tubes, 1.7 mL
- DNA Samples
- 6x Loading Dye
- Micro Pipettes
- Microcentrifuge
- Power Supply
- Ethidium Bromide
- Gel Photo Imaging System
Procedure:
Lab 4a:
- Calculate amount (mass) of NaCl needed to make a 5 M NaCl solution.
- Put said amount of NaCl into 15 mL test tube, fill with water up to 10 mL mark.
- Label test tube and store for later
- Calculate amount of TRIS and EDTA to make TE buffer
- Pour reagents into 250 mL beaker
- Add 80 mL of de-ionized water to beaker
- Add Sodium Hydroxide or Hydrochloric Acid to adjust pH
- QS to 100 mL
- Label and store for later
Lab 4b:
- Dilute DNA with TE in a beaker, calculated with (Concentration1)(Volume1) = (Concentration2)(Volume2). Record observations
- Add 500 microliters of 5 M NaCl
- Add 4 ml of ETOH to the solution by trickling it down the side of the beaker
- Spool the DNA with a glass rod
- Label and store for later
Lab 4i:
- Calculate how much agarose is needed to make TAE
- Add agarose to 100 mL of 1x TAE
- Heat mixture in microwave until boiling. Let cool
- Pour solution into a gel box with comb that has the open ends taped off
- Label and store for later
Lab 4j:
- Remove tape and comb from gel box
- Place gel box into gel tank, submerge in TAE
- Put 2 microliters of the DNA and 4 microliters of 6x loading dye in a 1.7 ml tube, run for 2 seconds in mini centrifuge
- Use a micropipet to transfer mixture into wells in the gel, making sure not to put in air bubbles
- Plug in gel tank to power source at 110 V for 45 minutes
- Remove gel and place into new gel box, stain with ethidium bromide overnight
- Rinse off gel, observe under UV light.
Data Analysis:
In actuality, our experiment wasn't a success. We followed all of the steps up until #7, since our DNA somehow wasn't showing up after we stained them overnight. Instead, we had to come up with possible problems and solutions. Since everyone had the same result, we could rule out individual errors and doing the procedure wrong. It had to be something that was universal. The reagents maybe? They all seemed to react as they were supposed to, the stain was the only problem. Ah, then it could be the ethidium bromide. Our teacher had used the same stock from last year, assuming that it could be stored indefinitely. But, we don't know if being exposed to sunlight or something else could have corrupted it. So, we should make a new batch of ethidium bromide and test it.
Update: After restaining the gel, we were able to get tangible results, as shown in the picture below.
Conclusion:
The process of isolating and purifying DNA on a gel is very valuable to anyone who has to deal with genetics or DNA. DNA can only be read if it is purified, otherwise it is like reading a book with coffee spilled on it, it is impossible. Once a scientist has purified the DNA, they can manipulate it however they wish. Forensic scientists can use it to match with the sequences in their database, and find people who were at a crime and possibly a suspect. Anyone trying to make a hybrid life-form could use purified DNA. It is the building block of who we are. The uses for it are limitless.
Reflection:
Our group's philosophy was: the more, the less work for each. With six people, we got everything done really fast and without a large amount of effort from any one or two people. With one or two people doing small things, we were done with the current task with lots of time to spare, all six of us. If that's not efficient, I don't know what is. The only downside to this fact was that if one member of our group messed up, it hung up a lot of people. Since we were all supposed to make a DNA-Loading Dye mixture and then inject it into the gel, when one member of our group had to do the whole thing over we were delayed a whole period. Other than that though, it was a fun lab that was made even better by a good, efficient group. As for the delay, practice will make (near) perfect.