Purpose A: To make 10 mL of 5m NaCl Solution and to make 100 mL of TE buffer out of 10 mM TRIS and 1 mM EDTA. Purpose B: To describe the properties of the DNA, if it can be spooled, and how much can be recovered from the solution. Purpose I: Make and pour agarose gels for DNA fragment analysis. Purpose J: Describe the appearance of different DNA samples on an agarose gel.
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 None of our DNA showed up in either of the two STEM classes, so this lead us to believe there was a problem with something like the stain. The DNA shouldn't have diffused over night, it was too big; even if the DNA had denatured, it would still stain; we could have not loaded correctly, or not made the buffers right, or not resuspended the DNA before loading, but it's not likely to happen to everybody; one of the reagents could have been bad, the stain is light sensitive and could have broken down over time. To test if the stain is good or bad, we can remake 1X solution from the old 20,000X stock, or we can create a new batch of 20,000X stock and dilute it again to 1X, which is what Dr. LB did. She stained one gel with each new solution and waited a few hours to see if the stain was the culprit.
In the end, when she made a new solution from new stock and stained the gels, the DNA showed up fine. You can see it in our picture above.
Conclusion You could use the DNA that you isolated to use in tests that specifically use pure DNA.
Reflection Our group worked together really well. We didn't fight and were pretty efficient, although pretty slow and calculated. We did make a few mistakes, mostly in practicing how to use the micropipettes, but the lab was very straight forward. We now know how to use the micropipettes properly and well, so that shouldn't be a problem in the future. As for making solutions and adjusting the pH of a solution, we learned quite a bit. We learned about buffers (and how to make one), and that if the pH of a solution is too high or low to just add an acid or a base to adjust it. I think we should definitely practice making solutions more, they are pretty essential to most experiments and we will probably end up making them a whole lot anyways.