Telomere science project

[carolinethorn]

Hi Eric,

Yes, the times used for digests vary. Some of this is because digests are used for different purposes. Sometimes you do a digest as a quick test that a particular fragment is present in your test sample, sometimes to prepare a piece of DNA to ligate into a plasmid and clone it. Different uses require that only some of the DNA is cut (as above) but some uses you want to be sure that all the copies that can cut, are cut - usually for trying to quantify the fragment, and then you would do a longer digestion (anything from 2hours to overnight).

So what do you think?

-Caroline

PS- Don't forget to mix the digest after you add the DNA in. (I would close eppendorf tube, flick a few times then give a quick pulse spin in the microcentrifuge to get everything down the sides so its all in the mix)

[ericjang]

Hi Caroline,

The statement you made seems completely reasonable- I guess I will leave the samples in the water bath for an hour then to make sure the enzymes have a chance to bind to the DNA. It should not be a very large problem, and patience prevails over shortcuts in the long run. Thank you so much for the mixing idea! I had never known that before. Should I incubate the tubes first or pulse them in the microfuge first before running the gel?

I recently just acquired my kit from SCCBEP. Over the past three days, I have been conducting mini-experiment tests to see if all of the equipment works. I am quite pleased to say that everything is going along as planned, save for three things. I am not sure of the level of problematic "magnitude" they have, but they do pose a good degree of hinderance. Fortunately, they all are things that seem to be easily fixable.

The first problem I encountered was that I grew one dish of yeast in yeast growth agar overnight. Yesterday, I prepared an 85 F degree water tank, filled a dish half full with hot agar, and then let it sit until it solidified and was stiff to the touch. I then sprinkled some yeast over in three areas on the dish. I set the dish directly into my aquarium environment (I did a quick weight test to see if 3.45 grams of petri dish could float in water), and let it float there over night.

The next morning, I was quite shocked to discover that the yeast had made no significant growth at all (In fact, I have pictures of the yeast from that show that when one is rotated, they almost look exactly the same. One is attached with this post, the second will come right after). Hypothetically, if the temperature was just right and the aquarium was functional, the yeast should have doubled approximately doubled in size 8 times (1.5 every hour at optimum temperature). I don't know if whether I killed the yeast, or whether the aquarium heat failed to warm the dish through conduction. Do you have any insight to what might have happened?

Another big problem that both me and 3 of my science teachers were unable to explain is that overnight, almost all of yeast agar mysteriously vanished. Today I was astonished to find that a very, very thin layer of agar was left under the yeast. I haven't the slightest lead on what could have happened, but I know it was probably a mistake on my part. Since the agar already solidified, I don't think it was possible for it to evaporate. The only explanation I can think of is that it could have somehow leaked out of a dish and mixed with my aquarium environment. However, this does not seem very likely as the dishes are quite airtight. The same question I asked in the previous paragraph, can you explain what happened? This is the problem that has especially left me perplexed.

Finally, I ran a test with my water bath to see whether it can incubate water at 37 degrees Celsius. When I turned the water bath on, a small pilot light switched on, but about 10 seconds later, immediately switched off. Another 10 seconds later, the light turned on again. I haven't had the time yet to run the bath for an extended period of time, but I did not notice any bubbles while the bath was heating. Again, the same question: Is there something wrong I am doing or is the bath supposed to do that?

Thank you for clarifying so many of my questions!

Eric Jang

[ericjang]

Hi Caroline,
Here is the second photo(the system won't let me paste multiple attachments or anything over a certain file size)

If you rotate the file about 45-80 degrees, you will notice that the pictures look nearly exactly the same, except the "day 1" picture has A LOT more yeast growth medium inside of it, as shown by the tannish color

Thanks!
Eric Jang

[carolinethorn]

Hi Eric,

Well done for thinking to test out your equipment before the big experiment.

Lots of questions within questions in your email. I will try and go through each one.

1. Lack of visible growth.
Maybe your starting yeast is dead?
I am a bit confused when you said you "sprinkled" the yeast. What kind of yeast are you using? In general when trying to grow a plate of yeast you start from a liquid culture and pipette the solution on and spread it with a sterile spreader or you can transfer yeast from another plate with a loop.

2. Disappearing media.
This is a weird one. When you leave plates for a very long time (months) the agar does tend to shrink but to do so in a couple of days is odd.
I have never tried to grow plates in a water based temperature environment though. I would be concerned that water is getting in somehow. The plates have to have some way to allow gases in and out so perhaps the agar is getting wet and dissolving and leaking out. which way up are the plates? big plate on bottom and smaller one with media upside down on top ?

3. Can you get a simple thermometer to test the water bath? Some of them the light only comes on when the heating element is on, when the water gets to the set temperature the light turns off. Hopefully this is the case but it would be good to check.

I'm sorry i don't have better answers. I suggest you try making a new thread and titling it something like "problems with growing yeast" to discuss your yeast and media issues. People who know about yeast might be more likely to see it and reply than to this thread.

best of luck,
Caroline

[ericjang]

Hi Caroline,

Thank you for your answers! I will also address them one by one to avoid confusion.

lack of visible growth
1.) Well, when I grew the yeast a couple days ago I had a jar of ordinary baker's yeast. After the agar in the dish solidified, I pinched some dry yeast out of the jar and then sprinkled it on top of the agar. It is a possibility that the yeast could be dead, but on the sciencebuddies bacterial troubleshooting guide, I discovered that some fungi, especially bakers yeast, takes 3-5 days to start dividing rapidly, so that may be the case. I haven't tried growing the yeast in a liquid culture first, so maybe I should try that out- packaged yeast comes vacuum-packed in stasis mode with a coating of dead cells around it, so maybe the layers have to be dissolved first so the nutrients can get through.

disappearing media
2.) The petri dishes I have are taller dishes with a smaller diameter with lids that are shorter but have a larger diameter (I figured that it wouldn't make sense to have a taller lid than dish, so I assumed that the shorter but "wider" dishes were lids). I wanted to grow the yeast aerobically, so I left the lids off while they were in their water environment. I was suspecting that the dish might be slightly porous, so on Friday, I left 4 more dishes out to see what would happen. One of the dishes was the exact same as the one I did previously, with no lid. I prepared another with a lid on it, as well as a dish with a 3g weight in it and another dish with water in it. I wanted to see if some water would enter the dish over the weekend and at the same time determine if the yeast would grow. But at the same time, it could be because I didn't grow the yeast in a liquid culture first

3.) I'll be sure to run a test with a thermometer


Thanks!
Eric

[carolinethorn]

Hi Eric,
How did those last tests go?
I didn't see, did you try making a new post to get the attention of the yeast experts?
best of luck,
Caroline

[ericjang]

Hi Caroline!

I ran some tests with the water heater and more tests with the disappearing agar. The water heater works fine, except it has a problem of slowing warming up, since it is not thermostat regulated. After a week's worth of agar testing in a multitude of environments, I have modified my setup so that the dishes are enclosed in a container that is then submerged into the tank of water. This way the heat is distributed properly, but the water will not get in. However, even after transferring the cells from liquid media into the dish, it appears that yeast still is not growing. Since yeast is such a troublesome experiment organism with all the conditions it requires, I was thinking maybe I could switch my model organism in my experiment. I will be conducting some research as to how I should adapt my procedure correctly.

Sorry, I forgot make a separate thread. Thank you for reminding me!

Eric Jang

[ericjang]

Hi once again,

For the last couple weeks, I have been posting some of my other problems on separate threads. However, recently I feel that it might be a good idea to post a new problem of mine on this topic because it is related to the restriction enzyme digestion protocol.
I am a little bit confused with the reagents I must combine for a HindIII restriction digest:

I currently have HindIII enzymes, distilled water, and 5x enzyme buffer. My procedure, as following the protocol mentioned on this site: http://www.methodbook.net/dna/restrdig.html,
combines 2 ul DNA, 6.5 ul water, 1 ul buffer, and finally, 0.5 ul of the enzyme (In my experiment, I am doubling the volumes of each reagent to maintain the same ratios). However, this procedure uses 10x buffer in its calculations.

My main concern is that since the buffer is at a 5x concentration, would I have to dilute it? I have received notice that I must dilute to a 1x concentration before use, but since the protocol on the aforementioned website already adds 13 ul water to the 10x buffer, ideally, the buffer would be diluted to about 1.3x. So if the buffer is indeed diluted by the water, I would have to add 8ul of water instead to dilute the buffer concentration to 1x?

I am sorry if this causes any confusion: I have a very limited supply of reagents and I am concerned that I will be running out of materials if I budget my concentration volumes poorly. Also, it probably causes problems with the results of the actual experiment as well.

Thanks!
Eric Jang

[ericjang]

Hi Everybody,

I just wanted to thank everyone for all the help I have received from the kind experts! I was able to pull of my experiment (5 days before the fair). Although it was close, I got all the troubleshooting issues fixed!
The science fair went by very well!


Thank you all for your generous support!
Eric Jang
Cupertino High School

[amyc]

Hi - I am glad you were able to get everything wrapped up and together in time for the science fair! You were competing at the Synopsys Science Fair Challenge, right? I know you said it went pretty well. Did you get positive feedback/response from the judges?

Amy
Science Buddies

[ericjang]

Hi Amy,

Yes, I did participate in the Synopsys challenge. Much to my dismay, it slipped my mind to get constructive feedback about my project from the judges However, everyone I met was very nice and enthusiastic about listening to what I had to say.

Thanks!
Eric

[ericjang]

Hi all,

I would like to thank you all for your support towards my telomere science project! I managed to pull off a 1st place award at the Synopsys Science Fair. Unfortunately, I did not qualify to participate in the Intel Science fair- however, I was invited to participate in the California State Science Fair, in which I received an honorable mention from the Health Physics Society for my research in the dangers of Ultraviolet Radiation. I also discovered that I am the first student from the 50-year history of my high school to ever participate in the science fair (pretty neat!)

This year, my greatest regret was that I was unable to find a lab to conduct my experiment in- I intend to partake in a much more advanced project (although probably in the same field of research) next year. Consequently, I will definitely need assistance from a biotechnology lab- If anybody has any contact information for finding a mentor (yes, I have already read the 'How to Find a Mentor' link), I would greatly appreciate it.

Once again, thank you all for your generous support!
Sincerely,
Eric Jang

[amyc]

Erica - Congratulations on your win at Synopsys and on the honorable mention at the California State Science Fair. That's wonderful, and it sounds like you are already think about next year!

Amy
Science Buddies