-Introduction: Before I start talking Labs 4a, 4b, 4i, and 4j, my group (Rick, Max, Cris, Garrett, Toby, Brain) and I had to make a solution for DNA isolation and analysis the results. Below is the process, materials, procedure, data, conclusion, and reflection of this entire experiment. There is a lot of information, so be prepared to read for a while. -Purpose: 1.) Make 10 milliliter (mL) of 5 M NaCl solution. 2.) Make 100 mL of TE buffer: 10mM TRIS, 1 mM EDTA (DNA storage solution) -Materials: ~Lab 4a, 4b, 4i, & 4j: - Balance, analytical - Balance, tabletop milligram - Weigh paper, 7.6cm x 7.6cm - Weigh boat, 3.5" x 3.5" - Lab scoops - Sodium chloride - Tubes, 15 mL, capped - Tube racks for 15 mL - TRIS - EDTA, disodium salt - Bottle, 12.5 mL - Graduated cylinder, 100 mL - pH paper, wide narrow-range - Hydroelectric acid - Sodium hydroxide - Glass rods - Filtering flasks 250 mL, 0.2 um (micro-milliliter) - Vacuum pump and "trap" jar - Sodium chloride - Beakers, 50 mL - Micropipet tips for P-1000 - Permanent lab marker pens - DNA salmon testes - Ethanol, 95% - Plastic beaker, 1L tripour - Pipet, 2 mL - Glass rods - Pipet pump, blue - Micropipet, P-1000 - Tube racks for 15 mL tubes - TAE buffer concentrate, 40x
- lab scoops - Safety glasses - Beakers, 600 mL - Media bottle, 250 mL - Gel box, horizontal, for agarose gels - Balance, tabletop milligram - Agarose - Microwave oven - Weigh boat, 3.5"x3.5" - Water bath, 65*C - hot hands protector - pBR322, 50 ug/uL and loading dye - Lambda/HindIII, 50 ug/uL and dye - prepared agarose gel - Lambda DNA, 50 ug/uL and loading dye - Power supply - Ethidium bromide, 0.5 ug/mL - Tube rack for 1.7 mL tubes - other DNA samples and loading dye - Gel photo imaging system - Thermal paper - Reaction tubes, 1.7 mL - Gel loading dye, 10x or 6x - Thermal printer - Micropipet, P-100 - Micropipet, P-10 - Large gloves - Miropipet tips for P-100 - Micropipet tips for P-10 - Microcentrifuge - Yeast DNA, 50 ug/uL and loading dye - Weigh boat, 5.5"x5.5"
-Why and What...? - Why NaCl?A: Positive charges of Na+ bind DNA so it can clump together and form a precipitate. - What is Precipitation?A: Taking something out of a solution - Why TRIS?A: Maintains an pH of 7-8 - Why EDTA?A: prevents DNase activity - What is DNases?A: Enzymes that breaks down DNA -Process: In this experiment, my group and I received Salmon sperm and we spooled and dried it using TAE and other stuff. We then made a conductive gel that we put the DNA into so we could see how it moved and/or un-spooled as we sent an electric current through it. We took a picture of our results because the UV rays would hurt our eyes. Below are the steps and notes we took for each Lab. -Lab 4a: ~Solution 1, 5M NaCl: -Step 1: First, my group and I had to determine the amount of NaCl we needed. We used the formula M(Mole concentration) * V(Volume) * FW(Formula Weight). Using this Formula, we found that (5M)(0.010L)(58.44) = 2.92g NaCl
-Step 2: Next we used the analytical scale to carefully measure 2.92 grams using he weigh paper.
-Step 3: We then put that into a test tube and poured de-ionized water up to 15ml in the test tube, mixing them with the vortex machine.
~Solution 2 TE Buffer: -Step 1:Determine the amount of TRIS and EDTA for TE Buffer using formula M * V * FW
-Step 4: Being even more careful and precise than last time we used the analytical scale to get the amounts and put them into the same 125ml tube. Then we filled the tube up to 100ml and mixed them well.
-Lab 4b: -Step 1: Dilute DNA with TE in beaker. Once Diluted, observe.
-Step 2: Add NaCl 500 mL
-Step 3: Add 4 ml EtOH (trickle downside)
-Step 4: Spool DNA
-Step 5: Put PNA into new tube with 2ml fresh TE and Label -Lab 4i: -1x TAE: ~Make from 40x stock ~C1V1 = C2V2 --> V1 = C2V2 / C1 ~C1 = Stock concentration ~V1 = ? ~C2 = Final Concentration ~V2 = Final Volume ~V1 = (1) * (500ml) / 40xTAE = 12.5ml
-Step 3: Heat to boil & dissolve (heat - swirl - heat - swirl until clear)
-Step 4: Let cool until you can touch flask for few seconds -Step 5: Pour in prepared mold & let cool
-Lab 4j: -Step 1: Remove tape from gel and place in gel tank. One half of the tank is Negative/Black and another Positive/Red.
-Step 2: Pour TAE over gel until covered. Then gently remove combs
-Step 3: Prepare samples: ~20l DNA + 4ul 6x loading dye ~Spin 2 sec. in mini centrifuge -Step 4: Load samples onto gel p20-200
-Step 5: Put cover on gel tank, plug into power supply
-Step 6: Run at 100v for about 45 minutes
-Step 7: Stain several hrs. with EtBr (Ethidium Bromide); rinse & observe with UV light
-Data Analysis: The next day, my group and I were going to check to see the DNA change, but we ended up not see them. Instead, the gel appeared clear with purplish dots and they were all about the same distance from the beginning position before the electricity ran though them. Each person in my group gave us a total of seven dots. There could be many reasons for the lab to have not produced a DNA band such as. Most likely though, the EtBr dye probably corroded during the process so we can't see the band, even though it is likely there.
-Conclusion: I found this over all interesting and can be helpful for future research of DNA samples. This information can also give feedback on things we did correctly that could help other failed tests or show how something went wrong compared to something that went right.
-Reflection: As said in the conclusion, I found this experiment interesting, but also very hard. It was really hard to understand all these concepts and do it in such a short amount of time. Plus we had SEVEN people in our group. That's way too much. I need to be in a smaller more controlled group next time because seven people is ridiculous and a lot of us didn't have a lot to do. Although we didn't have a lot to do, work could have been separated better to make this project more effective and easier to control. When my class does a lab again, I want to try to find a more controlled group, not trying to be mean to my friends but they distract me and everyone. Hopeful it works out better next time :-\