10/8/14
Purpose- 10 mL of 5 NaCl solution, to make 100mL of TE buffer: 10 mM TRIS, 1 mM EDTA (DNA storage solution), to find out if DNA an be spooled out of solution, to find out what DNA looks like, to find out its many unique properties, to find out what yield of DNA can be recovered during the isolation, to prepare and pour an agarose gel for DNA fragment analysis, to find out what the appearance of different DNA samples on an agarose gel.
Materials
Procedure
Day 1
Data Analysis
Sadly, the DNA bands did not end up showing up. When discussing with the rest of the class, we put together a list of possible reasons for the experiment going wrong and how likely they were to have happened. After, we came to a conclusion that we had done something wrong with the stains when we made them. We discussed that this is very likely because it was hard to load exactly and could have broken down from the UV rays over the past year.
Dr. LB changed this to redo this lab with a new stock solution.
Conclusion
Isolating the DNA from the solution is a big part. It is a very complex step, but can be used for many reasons. Scientists can analyze DNA when it is purified by using the gel as a medium for the DNA. A gel can suppress thermal convection and delay the passage of molecules. This can help a scientist be able to study an individual gene and see if it contains a disease. If so, it can help a scientist produce a cure for a disease or something to help it.
Reflection
This project was very interesting because there was so many steps. My group worked surprisingly well. I was not there the day we started to work in our lab and chose our group, so I went to one with few people. We cooperated very nicely and were able to get things done quickly with being able to get it thoroughly. Although, our group added too much water to the concentration, so we combined with another group to share solutions and materials with them. Also, I could work on being able to use the equipment better. I was starting to get the pipetting down, but once we went to smaller pipets, I was not as smooth with releasing. Overall, this was a great lab and really taught me you have to pay close attention to what you are doing because it may not turn out as you expected.
Purpose- 10 mL of 5 NaCl solution, to make 100mL of TE buffer: 10 mM TRIS, 1 mM EDTA (DNA storage solution), to find out if DNA an be spooled out of solution, to find out what DNA looks like, to find out its many unique properties, to find out what yield of DNA can be recovered during the isolation, to prepare and pour an agarose gel for DNA fragment analysis, to find out what the appearance of different DNA samples on an agarose gel.
Materials
- balance (analytical)
- balance (tabletop milligram)
- weigh paper
- weigh boat
- lab scoops
- sodium chloride
- tube racks
- tubes
- TRIS
- EDTA
- bottle, 125 mL
- graduated cylinder, 100mL
- pH paper
- hydrochloric acid
- sodium hydroxide
- glass rods
- beakers, 50 mL
- DNA salmon testes
- pipet, 2mL
- pipet pump, blue
- micropipet, p-1000
- micropipet tips for p-1000
- ethanol, 95%
- lab marker pens
- plastic beaker
- TAE buffer concentrate, 40x
- beakers, 600mL
- agarose
- media bottle, 250mL
- microwave oven
- hot hands protector
- gel box
- beakers, 50mL
- water bath, 65 degrees Celsius
- tube rack for 1.7mL tubes
- reaction tubes, 1.7mL
- gel loading dye
- micropipet (p-10)
- micropipet (p-100)
- microcentrifuge
- power supply
- ethidium bromide
- gel photo imaging system
- paper (thermal)
- printer (thermal)
- gloves
- glasses (safety and plastic)
Procedure
Day 1
- weigh the sodium
- put 10mL deionized water into 15mL test tube w/ 2.92g sodium
- vortex
- weigh EDTA of 0.37g
- weigh Tris for 1.58g
- add Tris and EDTA & 80mL water into a test tube to make 100mL TE
- record pH level
- raise pH to 8 w/ base
- add sodium hydroxide to adjust pH
- put 1mL TE buffer w/ salmon sperm sample
- w/ keeping everything chilled
- add 500 microliters
- add 5 ml ethanol
- spool & observe DNA
- put DNA into test tube
- make 500mL of 1 x TAE buffer from 40 x concentrate
- weigh 0.4g agarose
- heat flask to dissolve
- prep gel mold
- pour gel into gel mold & let cool
- remove tape from gel, place in gel tank
- pour TAE over gel until covered; gently remove combs
- prepare samples
- load samples onto gel
- put cover on gel tank, plug into power supply
- run at 110v for 45 mins
- stain several hours w/ EtBr; rinse and observe w/o light
Data Analysis
Sadly, the DNA bands did not end up showing up. When discussing with the rest of the class, we put together a list of possible reasons for the experiment going wrong and how likely they were to have happened. After, we came to a conclusion that we had done something wrong with the stains when we made them. We discussed that this is very likely because it was hard to load exactly and could have broken down from the UV rays over the past year.
Dr. LB changed this to redo this lab with a new stock solution.
Conclusion
Isolating the DNA from the solution is a big part. It is a very complex step, but can be used for many reasons. Scientists can analyze DNA when it is purified by using the gel as a medium for the DNA. A gel can suppress thermal convection and delay the passage of molecules. This can help a scientist be able to study an individual gene and see if it contains a disease. If so, it can help a scientist produce a cure for a disease or something to help it.
Reflection
This project was very interesting because there was so many steps. My group worked surprisingly well. I was not there the day we started to work in our lab and chose our group, so I went to one with few people. We cooperated very nicely and were able to get things done quickly with being able to get it thoroughly. Although, our group added too much water to the concentration, so we combined with another group to share solutions and materials with them. Also, I could work on being able to use the equipment better. I was starting to get the pipetting down, but once we went to smaller pipets, I was not as smooth with releasing. Overall, this was a great lab and really taught me you have to pay close attention to what you are doing because it may not turn out as you expected.