Telomere science project

[carolinethorn]

Wow Eric, that is very inventive!
It is a somewhat unconventional method so you might want to think about how it may effect your yeast. Generally, culturing cells over 10 days you would want a way to remove a lot of the dead ones so they don't release growth inhibitors and toxins into the media. This can be done by sub-culturing, taking a portion of your old culture and putting it into new medium. This might be important when you start hitting them with UV and killing a bunch of them.
You also said you are growing them in sugar water but they might need some other nutrients in there to aid growth so it might be worth looking into yeast growth media. As I said before I am not a yeast expert, so i am not sure about this, but other organisms need to get a few essential amino acids from external sources.

About your HindIII question. There should be a way to get a HinDIII restriction map for the Saccharomyces cerevisiae genome. Perhaps at SGD the yeast genome database, www.yeastgenome.org/.
sounds like you are doing great with your preliminary work,
-Caroline

[ericjang]

Dear Caroline,

Thanks for the input! I have a question about the subculture protocol though. How would I go about transferring the "good" yeast cells to a new growth medium? I am a little worried that if I take a random sample from my original culture, that might reduce the population significantly (They are only exposed to optimum temperature 4 hours a day, so at the maximum, they are only able to double in size a about 5-6 times in a 2 day gap, and given natural causes, some may die off or be used for DNA extraction)

As for the growth media, I don't think the yeast requires any external nutrients, at least for my experiment. Although it is true that many organisms require nitrogen, the yeast should be okay- The fermentation rate was only about halved at the end of my mini-test cycle.

Thanks for the restriction enzyme link! I found inside this section http://www.yeastgenome.org/cgi-bin/seqTools
It can generate the sequences for ALL of the chromosomes. I used Ctrl-F to find where there was AAGCT, and discovered various places where the DNA gets cut. However, there are some "chunks" of DNA that appear to be quite large even after HindIII digestion (cuts only 64 times), so I am a little worried about how to find the telomere region if it isn't the largest string of DNA.

[carolinethorn]

Ah, they are growing very slowly. I guess you aren't accumulating as many dead i had i thought (I was thinking population doubling every hour like bacteria and so it would be very different after 10 days!).

[ericjang]

Yes, they are growing relatively slowly since they are exposed to their optimum division temperatures only 4 hours a day.

Meanwhile, I have contacted both SCCBEP and the Schmahl science workshop for asking if they have any of the resources I need, but so far, I have not received replies from them lately. Are there any other places where I can get electrophoresis equipment, restriction enzymes, and a micro centrifuge?

Thanks,
Eric

[carolinethorn]

Hi Eric,

I'm sorry I don't have any suggestions other than to try your contact at Stanford again and see if they can let you work a few hours in their lab. Microcentrifuges can cost a thousand dollars and so its unlikely your school could purchase one. It might be possible to borrow a gel box from a lab (they cost a couple of hundred dollars and most labs have a spare) but you will need a power source (your school's physics lab may have one). what kind of gel were you going to run? are do you have a source for the gel?
Restriction enzymes are in the $100 range but you probably wouldn't need a whole vial so a lab might let you have enough for your experiment. I don't know if you can buy these privately.
Do you have access to a large centrifuge? how were you planning on doing the DNA extraction from your cells?
there are so many pieces of equipment that we take for granted when working in a lab.

Hopefully someone at the SCCBEP or the Schmahl science workshop gets back to you with good news.
best wishes and happy holidays,
Caroline

[deleted-2131]

Hi Eric,

I noticed that you are located in the Bay Area (I gathered that since you are working with SCCBEP and the Schmahl Science Workshop. I assume that you are entering the Synopsys fair since you are working with Schmahl. In that case, it is highly likely that your teacher knows someone of the SCVSEFA committee. If your teacher can explain to someone on the committee what it is you need, it is highly likely that they can help find you the needed equipment. You can visit the Synopsys fair's website at science-fair.org. You can visit the Synopsys Outreach Foundation here: http://www.outreach-foundation.org/.

[ericjang]

Hi Terik,

I checked the website and according to the area that I live in (Santa Clara), they have already exhausted their grants for this year, as the deadline for application submission is past due.
Unfortunately, I don't think my teacher has any connections within the committee, but perhaps I can contact a member of the committee myself.

Thank you so much for the link!
Happy Holidays,
Eric Jang

[deleted-2131]

Hi Eric,

I would still go ahead and contact someone at the Synopsys Outreach Foundation explaining your problem and the equipment you need. They have the connections to the people who have that sort of equipment and can put you in touch with them. Click on the "Contact Us" link and send them an email. I''m also glad that you will be talking with someone on the SCVSEFA committee. They , too, have the connections with peope who have what you need to use.

Keep us posted!

[ericjang]

Thank you so much!

I managed to get in touch with somebody who can provide me with all the materials I need, except one. Although it is not of critical importance, my experiment could have much better results if I could get a spectrophotometer, as it can quantify DNA. Does anybody know by chance where I could possibly get one?

Meanwhile, my experiment is coming along fine, but I ran into a slight issue. I just switched my growth medium/container from a sugar solution in a flask to yeast growth agar in petri dishes. I previously knew how to take the cells from the liquid stock, but I couldn't find the protocol for removing the yeast cells from the petri dish. How would I go about extracting such cells? Also, according to several online protocols, I find it strange that before running a gel, one submerges the agarose in about 2-5 mm deep of electrophoresis buffer. If the gel is submerged, how does one load the DNA?

Happy new years,

Eric Jang

[carolinethorn]

Hi Eric,

Glad things are moving along well. Sorry, I can't help on the equipment side.

As for harvesting the cells, it should be possible to use an inoculating loop to scrape them up from the agar plate. Here is a description for bacteria (see rapid colony transformation)
http://www.accessexcellence.org/AE/AEPC ... otypic.php
We also used to use sterilized flat toothpicks but you would need to autoclave those to sterilize them, an inoculating loop you could sterilize in a bunsen flame. Or you might be able to buy pre-sterilized plastic scrapers.

Loading a gel can be tricky the first few times. If you can practice first with just some dye solution that would be a good idea. Here is a diagram showing how you put the pipette tip through the buffer into the well (scroll down pretty far). The dye has glycerol in it to weight down the DNA so it stays in the well long enough to start the current and get it into the gel.
http://ocw.mit.edu/OcwWeb/Biological-En ... mod1_2.htm

Hoe this helps. Happy new year,
Caroline

[ericjang]

Hi Caroline,

Thanks for the info on the inoculating loop! I'll try to get one of those if possible, as it seems that they can be reused over and over again. If not, would it be possible to use a common kitchen utensil, such as the back of a plastic knife to scrape the cells off the dish?

Thank you for the input on the method of loading the gel! I was beginning to get worried that something was wrong with my procedure since there was a layer of liquid on top of the gel right before the loading step. But the loading buffer(marker dye) will act as a weight and sink the DNA to the bottom of the gel, right?

Having no prior experience in gel electrophoresis, about how long would it take to run a 0.7% gel on 5ul yeast RE DNA per well? I would guess a couple hours, but I just want to ballpark the amount of time necessary so I can budget my time better.

Thanks,
Eric Jang

[carolinethorn]

Hi Eric,

It should really be a sterile utensil to transfer the cells from the plate. But i guess since you are transferring them to extract the DNA straight away a few stray bacteria shouldn't matter too much (like it would matter if you were streaking the yeast onto another plate to subculture). You could sterilize a metal knife or metal spoon handle in a flame though and use that. Let it cool down a minute though before you scrape with it, like it says for the loop.

Yes, thats right the loading buffer/marker dye will help keep the DNA in the well for a few minutes, long enough to load the gel, but then you need to start the current so the DNA gets moved into the gel. Once its gone into the gel a decent way you can even stop the current and take a look and restart and so on. The rate that the DNA will move through the gel will depend on how much current you apply, the size of the bands you are looking for and the percentage gel (there is probably an equation somewhere you can find to estimate from). 0.7% is a fairly low percentage gel so i think it will move fast, maybe even only take 30 minutes, so i think 2 hours is reasonable time to budget.

-Caroline

[ericjang]

Hi Caroline,

Thanks! Glad to hear that I won't have to stay up all night waiting for molecular weight dye to cross the finish line
I have never done very much advanced DNA extraction, and my experiment procedure worries me a little that the DNA might not come out right. I was only able to find one site with instructions for simple yeast spooling, and am concerned that the proportions might be off (there seems to be an awful lot of water ) Would anybody mind taking a look at my following extraction procedure to see if it is fundamentally flawed or lacking in a certain material? The reason to why there is such a small amount of yeast and chemicals is because I don't think I will be able to harvest that much from each individual petri dish. Thank!

detergent/water solution = 180 ml water, 20 ml detergent ratio
tenderizer solution= 5g tenderizer, 95 ml water ratio
1.) Remove 5 ml of yeast from each dish by scraping off a thin layer of cells from each dish with the plastic scraper. Place these in separate plastic bags. (For the first extraction, mix 5 ml of water with each 5 ml of yeast directly from the jar)
2.) For each of the 12 batches, Add 5 ml detergent/water solution.
3.) Mash each bag thoroughly for 4 minutes
4.) Pour mixture into a beaker, add 2 ml of meat tenderizer/water solution, and stir to mix. Add one milligram of salt and continue mixing.
5.) Place 1.5 ml of mixture into a microfuge tube.
6.) Spin the mixture in the microfuge for 2 minutes, with a tube of water on the opposite end as a counterbalance.
7.) Transfer the lighter layer of DNA into a new tube, and discard the protein precipitate.
8.) Pour about 1.5 ml of ice-cold ethanol carefully down the side of the tube to form a layer. It should double the volume of the solution. Invert a couple times to mix the solution.
9.) Spin the microfuge tube in the microfuge for another 2 minutes so that the DNA precipitates at the bottom of the tube.

electrophoresis

1.) Add restriction enzymes to DNA samples to break up DNA into fragments. (2µl 10x Buffer, 13µl H2O, 4µl DNA, 1µl Enzyme in a microfuge tube). For mass-digestion, prepare one large batch of buffer, water, and enzyme by mixing ingredients together and mixing by using the pipette to suck up and down (when you put the enzyme in after the first two ingredients). Then, distribute the solutions into tubes and with new tips, add the DNA on top of that. Incubate in 37°C water for about an hour.
2.) Meanwhile, boil a agarose solution (1 gram) in 100 ml of electrophoresis buffer in a microwave oven for one minute. Let solution cool to about 60 degrees Celsius. Stir the solution while cooling. Pour into gel tank.
3.)When the gel has cooled, (at least 30 minutes), carefully remove the comb.
4.) Pour in some of the same electrophoresis buffer you used earlier. It should submerge the gel to about 2-5 mm in depth.
5.) Add loading buffer (marker dye) to each of the DNA samples so that it is 20% of the DNA.
6.) Fill the wells in the gel with a sample of cut up DNA from each extraction. Switch on the power supply to about 5V/cm (distance between electrodes). The DNA should start to move to the positively charged end of the gel. Turn it off when the marker dye reaches ¾ the end of the gel.


Thank you guys so much! The last thing I want in my complicated experiment is to have too much water in my experiment procedure

[carolinethorn]

Hi Eric,

While my knowledge of the theory and processes in the lab still qualifies me as an expert, I haven't touched a pipette in about 5 years so I am probably not the best person to advise on your quantities problem but I do have a few pointers:
Does the protocol you found for yeast DNA spooling give an approximate yield in mg? This would help you figure out about the water issue. I don't think its a problem during the extraction procedure because at the end you get a pellet of DNA and can resuspend that in any volume of sterile water to do your digestions. The question is what is the appropriate volume of water! Most of the protocols that i found online that extracted DNA from a 3ml liquid culture resuspended in between 20-100 microlitres H20 or TE buffer. If you were to resuspend in 25 or 30 microlitres of water that would leave you enough to do your digestions and to spec a couple of microlitres to check concentration (make sure you do a quick pulse spin of the microcentrifuge tube of DNA before pipetting any out so you don't lose too much on the sides).
The second volume i think you could tweak is how much DNA you put into your digestion. The protocols that come with the enzyme usually state how many milligrams of DNA are digested per unit of enzyme. If you don't manage to get spectrophotometer access to estimate your DNA concentration, you could hedge your bets by doing 2 digestions per DNA sample. One with 4 microlitres of DNA as you described and one with 17 microlitres of DNA and omitting the added water. But then you would need twice as many lanes in the gel, or to do two gels. (NB: if you resuspend DNA pellet in 25 microlitres of water and something goes wrong with the gel or digestion that really doesn't leave enough left for another try but 30 microlitres would let you have a second chance at the 4 microlitre digestion- sometimes its worth budgeting for some errors when doing procedures for the first time!)

best wishes,
Caroline

[ericjang]

Thanks Caroline!

Your input on chemical quantity really helps my experiment- I will think about those suggestions for my experiment procedure.

I have been looking at several factors of my experiment in the past few days. I was reading the transition step between DNA extraction and the gel electrophoresis step, the part when I add the restriction enzyme solution to the DNA followed by incubation in a water bath for an hour. I am not sure whether an entire hour is necessary for the DNA. I read from a different source that I only need to wait for 45 minutes. As an expert, do you have any experience in how long you would need to wait for the digest?

1.) Add restriction enzymes to DNA samples to break up DNA into fragments. (2µl 10x Buffer, 13µl H2O, 4µl DNA, 1µl Enzyme in a microfuge tube). For mass-digestion, prepare one large batch of buffer, water, and enzyme by mixing ingredients together and mixing by using the pipette to suck up and down (when you put the enzyme in after the first two ingredients). Then, distribute the solutions into tubes and with new tips, add the DNA on top of that. Incubate in 37°C water for about an hour.

Thank you!
Eric Jang

[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