Week 9
Hello! I can't believe it's already week 9 of the project.
Anyway, the DNA ladder failed yet again on Friday, so we are letting the DNA and enzymes sit overnight. Hopefully that will be enough time for the enzymes to break up the DNA.
Today I helped prepare DNA samples for testing. First the lab manager collected tiny snips from the ends of the tails of the new mouse litter and put each in its own plastic tube. I added 20 microliters of NAOH to the tubes. This makes the solution basic, which partially dissolves the tail tips. After about an hour, I added a mildly acidic solution to balance out the pH. Then I put the tubes in the centrifuge so all the liquid would settle to the bottom, and stuck them in the freezer. Tomorrow we will put these samples in the gel electrophoresis tank and find out the genotypes of the mice.
Before going home I set put the DNA and enzyme mixture for the DNA ladder in the water bath so it will stay warm overnight. Hopefully it works this time!
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Wednesday update on the DNA ladder: it still doesn't work! :(
The DNA ladder still isn't working, so we ordered the restriction enzymes from a different manufacturer, because we think the first batch was defective. The new ones should arrive today and we will leave them overnight again. Hopefully they cut up the DNA this time!
Today I also got to autoclave some more pipette tips and do another electrophoresis to test the genotypes of some mice. I am also helping the lab manager make a protocol (set of instructions) on how to use the microscope with the fluorescent light. With the protocol it will be easier to teach new people coming to the lab, because there will be a detailed set of instructions on how to use it.
I'll make another update on the DNA ladder tomorrow, fingers crossed it won't be another failure!
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Hello!
We didn't get a chance to test the DNA ladder after waiting a whole night, but it did not work after the one hour test we did try. Probably the enzymes need to be left with the DNA overnight, so we will try that next week.
We will also be having a lab meeting on Monday, so I will give you an update on the information we talk about on Monday.
Have a nice weekend!
Hi Serena! I'm sorry to hear that the gel electrophoresis testing didn't work for you guys. It's high school biology all over again. So what sort of factors do you think might need to be changed for it to work in the future? Will you able to get it re-done and corrected during this last week?
ReplyDeleteThe lab manager called the company that supplies the enzymes and DNA used to make the DNA ladder and they suggested we use a different concentration of buffer. The water is turned off in the lab this week for a plumbing repair, so we will probably try that next week (I will still be coming in after the project is over).
DeleteHey Serena! Sorry that the DNA ladder isn't working out. I was wondering how the genotypes of these new mice will be compared with that of the old batch of mice. Thanks, and good luck!
ReplyDeleteEvery time there is a new litter of mice pups (about every other week) we test their genotypes to see if they are wild type, knockout, or heterozygous. We are not comparing them to last week's mice, just finding out what type they are so they can be sorted into different cages and used for different experiments.
DeleteHi Serena! I'm sorry to hear that you weren't able to test the DNA ladder this week. When you mentioned that the first batch of restriction enzymes were "defective", what exactly did you mean by this? I can't wait to read your last few posts! Good luck!
ReplyDeleteSophia Vaidya
We are not exactly sure what went wrong with the first restriction enzymes. All we know is that they didn't cut up the DNA, so we are assuming they are somehow defective.
DeleteHi Serena! Can you explain what a DNA ladder is? And why it's so frustratingly difficult to do? If the restriction enzymes really are defective, they must somehow have a hard time either recognizing the restriction site or cutting the plasmid up at the site... or something along those lines. It is difficult to believe it has already been nine weeks! I hope you have enjoyed your lab experience! What words of advice would you give to someone considering working in a lab? Thank you for the update! Only two more weeks to go!
ReplyDeleteThe DNA ladder is a known sample of lambda DNA cut at specific points by a restriction enzyme, mixed with a loading dye. As the gel runs, the dye moves along the gel, and allows us to see how much longer the gel must run. The ladder also serves as a control, as we can see in the finished picture of the gel that it does show all the bands it is meant to, meaning the gel electrophoresis worked correctly. We aren't sure why the restriction enzyme wasn't working, we have called the company that makes it and asked for their advice.
DeleteMy advice to anyone considering working in a lab is help out wherever you can. When I first started working here I mostly had little tasks to do like running the dishwasher, but now I am helping people with lots more of the day to day tasks around the lab.
Hey Serena,
ReplyDeleteI'm so sorry your DNA ladders did not work, but hopefully next week you wil have more effective trials. Could you explain how the gel electrophoresis would allow you to find out the mice's genotypes?
Have a great week!
Julie Losion
The tail tips from the mice are partially dissolved in a basic solution, and then mixed with a buffer to return the solution to a neutral pH. Then restriction enzymes are added. These cut the DNA at specific points based on the genes it has. If the mouse has different genes it will have different length pieces of DNA. The gel electrophoresis tank used a current to pull the DNA through the gel. Small pieces move faster, so at the end there will be distinct bands. Specific patterns of bands means the mouse has a certain genes.
DeleteHey Serena! How exactly do you prepare the DNA samples for testing? Also, why must you use the tiny snips from the end of the mouse tails to find the genotype? Hopefully next week the DNA Ladder will work!
ReplyDeleteAny tissue sample from the mouse would have some of its DNA in it. Tail snips are used because they are a relatively painless and simple way to collect a tissue sample from young mice. These tail tips are dissolved in a basic solution so the DNA can be extracted and cut up by restriction enzymes.
DeleteHi Serena! I was wondering how the preparation process (adding NAOH and a mildly acidic solution) helps determine the genotypes of the mice. Thank you!
ReplyDeleteThe basic solution dissolves the tissue sample so the DNA can be extracted. The slightly acidic buffer returns pH to neutral so it stop dissolving at a certain point.
Deletehey serena! I'm sorry to hear that the ladders are failing, but I'm sure that you will crack the code eventually. i was wondering do you test multiple versions of the ladders at the same time? keep up the great work.
ReplyDeleteNo, we try to only change one variable each time we test it so we can find out which one was causing it to fail.
DeleteHey Serena, I am sorry that the DNA ladder didn't work, but I hope it will work soon this week. How did u manage to measure 20 microliters of NaOH? What tool did you use?
ReplyDeleteA pipette
DeleteSorry for my late comment and to hear that the DNA ladders haven't been successful. I hope your new batch yields you better results! You've probably already had this question, but now that you've had more experience, what has been your favorite part of working in the lab? Thanks!
ReplyDeleteMy favorite part of working in the lab has been helping with actual research. Learning about all these concepts and techniques in Biology classes in school was interesting, but it is so cool to see how the actually apply to real world science.
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ReplyDelete