Telomere science project

[carolinethorn]

Hi Eric,
That is looking great. Good luck. I hope you get a positive reply.
Keep up the good work,
Caroline

[JinSoul]

From my knowledge of biology, I believe my AP biology teacher said there were current studies on how the lengthening of telomeres that could potentially help us extend our lives as well.

Also, to my knowledge, I believe that if we lengthen telomeres at an early start it may just increase the length of life because as we grow older our telomeres get shorter and shorter as they slowly get cut off. There is no definitive study that shows that telomere lengthening may be the key to life lengthening but there seems to be potential in the source. Originally during DNA replication telomerase is the enzyme that increases the length of our telomeres, so information on that could also lead to more information on your topic.

Here is a site that directs you to studies on the different methods of telomere lengthening:
http://www.futurepundit.com/archives/000664.html

http://www.nature.com/ncb/journal/v9/n1 ... b1664.html (explains differences in telomere length in male and females)

[ericjang]

Thanks for the cool info!


I have an additional technical thing that I have some issues with, concerning the T/S ratio.

I met with an expert in the area of biology, who currently works at Stanford. She told me that of all DNA assay methods, PCR is the fastest and cheapest way to conduct test- each only takes approximately 5 hours, and DNA samples can be frozen between experiments, which makes telomere assay via PCR probably the #1 candidate for measuring telomeres in my experiment. I have done a lot of research on telomere assay through PCR, of which is summarized along the lines of

"Mean telomere length and telomerase activity were measured quantitatively in PBMCs that were stored frozen at -80C. Telomere length values were measured from DNA by a quantitative PCR assay that determines the relative ration of telomere repeat copy number to single-copy gene number (T/S ratio)" (Blackburn 2003).

I am not very sure to what this means- could somebody please explain the T/S ration in a more "layman's" terms? I do understand the concept of PCR, which involves the mass-production of a certain gene through the use of taq polymerase and multiple cycles of heating/cooling. However, I do not see how telomere length can be "measured" in this way.

[carolinethorn]

Hi Eric,

That's great you have talked to an expert at Stanford. This is my educated guess at what the T/S is about but check back with your expert.

The telomere is a series of repeats of the same sequence. The longer the telomere the more repeat units. The PCR is designed to amplify the unit sequence. So if you do a quantitative PCR you can measure the number of unit sequences, then you would divide by the number of chromosomes to figure out how many units per telomere and so how long they are. But how do you know how many chromosomes you started with? That's where you use the single-copy number gene. It is a control. It should only amplify once chromosome per round of PCR so gives you a way to measure how many chromosomes you started with. So the T/S is a way of finding the relative length.

Hope this helps,
-Caroline

[ericjang]

Hi Caroline,

Thank you for your concise explanation. Not a single one out of the many journal articles I have read has explained the concept of PCR assay so well as you have

I have a couple questions though. In your response, you mentioned that "you use quantitative PCR to measure the unit sequences". I am not really sure what you mean by that, would you mind clarifying on what the difference is between quantitative PCR is and how it is used to measure unit sequences?

Also, I don't quite follow the concept of the single-copy number gene.

Sorry for the inconvinience



I have begun to flesh out my experiment. I have decided to do an experiment to find the increased telomere shortening in yeast when exposed to a stress vector. I have also figured the best way to conduct such an experiment was to have 3 groups: a control that is not affected, a test group that is exposed to an amount of the stress vector, and another group that is exposed to a larger amount of the stress vector. I have ruled out that temperature may not be a good idea because it can change the division rates of the yeast. I am not sure whether this may be important to the experiment, as telomeres are already known to progressively shorten with each division (thereby groups that have lower division rates will naturally have a longer average telomere length). I am considering sound to be a testable variable, although that requires the use of expensive soundproof containers and extremely loud speakers that must be kept on for days. Although sound may be a good independent variable, I am not sure if it is a good thing to test. Is there some other variable I can use in place of sound pressure that can induce some form of short-term physical damage while affecting cellular division rates as similar as possible, and at the same time, easy to apply?

[carolinethorn]

Hi Eric,

Your questions are smart questions and not at all inconvenient.

Quantitative PCR uses DNA bases that have a fluorescent probe attached. The quantitative PCR machine can measure how much fluorescence was incorporated and so how many copies of the unit sequence were made.

The single copy gene is a gene that occurs only once in the genome. I guess generally we assume most genes occur only once in the genome but actually thats not the case, there are some that are duplicated many times and for some the number of duplications can vary between different people. To be a good control it needs to be a sequence that occurs only once in the genome.

Sounds like sound might be tough
Other types of stress that are used in yeast that i can think of are cold stress and nutrient stress but i would think these would also effect the rate of division. Change in pressure or oxygen would require more specialist equipment i think. Might it be possible to have more test groups and try 2 groups for heat and and 2 for cold stress assuming then that they have opposite effects on division rates but are equally "stressful"? I don't know too much about yeast though.
It would be worth doing a literature search to see if these kinds of experiments have been done before. There are lots of stress response experiments that have been done, perhaps some have a good stress protocol but their end goal was measuring something else other than telomeres. You could then adapt their protocol for your needs.

Best of luck,
Caroline

[ericjang]

thank you for the clarification!

Application deadlines are coming up, so I am running out of time to submit my application. Working in a lab this year will probably not work out, so I will have to try to optimize my experiment so that it can be done at my high school. I have contacted SCCBEP, whom may be able to provide me with materials and resources. I need something that will be able to sequence the DNA so that I can measure telomeres- Which of the following kits would be best for me? Is it possible to compare telomere lengths by running a gel electrophoresis?

Here is the link to the SCCBEP resources website

http://www.science.sjsu.edu/SCCBEP/sche ... dules.html

[carolinethorn]

Hi Eric,

The method we were discussing before - quantitative PCR - requires a special quantitative PCR machine (sometimes called a Taqman machine which is a brand name). Quantitative PCR machines are much more expensive than regular PCR machines so you wouldn't get one for a high school lab. Plus the reagents (the fluorescent bases and primers) are expensive. These types of things are not in the kits from SCCBEP.
In theory you could do regular PCR and try and quantify the products from the intensity of the band on a gel (You wouldn't need to do "sequencing" as you don't need to know the bases present in the telomeres just the length). But measuring the intensity of the band on a gel requires a special type of digital scanner/camera and software (which i didnt see listed in the kits) and is not all that accurate. Given the difference in accuracy between trying to measure intensity on a gel and a quantitative PCR, I would think it would be hard to see any differences with your experimental conditions, but i might be wrong. It would be worth talking to your expert at Stanford to see if she can help suggest some ways to modify your experiment.

I am having a hard time coming up with an idea to adapt your experiment to a high school lab and still keep the experiments central goal to be about telomeres. The only things i can think of involve doing a bioinformatics experiment about telomeres. I am kind of biased because bioinformatics is my area of interest - using computers to look at previously collected data and do molecular biology experiments without a lab.

Best of luck,
Caroline

[ericjang]

Caroline, thanks for the consideration!

Although it isn't necessarily great that you had a hard time coming up with an idea to improve my project, I am very touched to hear that you spent time trying to figure out a way to make my experiment better/easier. My experiment, due to resource problems is stuck at the moment, but I am nonetheless glad that there are helping hands out there.

I have found an email exchange on the internet, of which I have posted a link below:
http://www.bio.net/bionet/mm/ageing/200 ... 04292.html
In that mail, I think it states that if you chop up the DNA with a restriction enzyme in the southern blot, it will not affect the telomeres anyway, and then that can be measured through gel electrophoresis by comparing the longest strands of DNA. Since the organisms I am experimenting with are the same species, any enzyme I use should leave the telomeres alone, and the lack of precision in separating the telomeres will not make much of a difference becasue the same will apply to the rest of the samples. I am not sure if this is true, but it is my presumption.

( I think the RSA/Hinf 1 enzymes are the restriction enzymes used for telomeres, as far as the link below says. http://cgn.pubhealth.ku.dk/upload/appli ... wborns.pdf )

However, you are correct that to measure the telomeres, I either need either PCR to amplify and then use a camera/scanner, or resort to southern blotting + probes. None of these are especially promising, as I either require expensive chemicals or hardware. There is an alternate method I am considering, called southern blotting. In normal Southern blotting, you basically run a gel of the DNA, and then you measure the intensity of a band by using a chromogenic dye or fluorescent probe. This is feasible to do with relatively simple equipment, save for it uses expensive chemicals. In addition, it requires many hours of waiting, in which I cannot be there 24/7 to replace paper towels, etc.

Caroline, I think the scanner+camera, although expensive, is the fastest and safest way to go. Is this link related to the kind of hardware you were talking about?
http://www.biocompare.com/Articles/Prod ... pment.html

Thanks,
Eric

[ericjang]

I think I have found some good news. I was conducting another 2-hour internet search today and stumbled upon this website
http://www.bio.net/bionet/mm/plant-ed/2 ... 07518.html

I think the person is talking about how you can do analyze gels by using a program called ImageJ and a standard camera. I downloaded the program and looked at some screenshots on the web, and it appears to me that it indeed has the capacity to measure band lengths, and therefore compare average telomere lengths. However, being an open source program, there are hundreds of plugins and macros online that I have no idea what they do and how they work. Could somebody please teach me step by step what I have to do to use this program to analyze a gel? (I am no computer whiz).

The link to the program's website is here, if it helps
http://rsbweb.nih.gov/ij/index.html

[ericjang]

I talked to my adult sponsor (my science teacher) and she says that in my experiment, to keep the temperatures in the yeast constant, I should use a heat source that does not produce UV radiation. I am not sure what kind I should use- an infrared heat lamp perhaps? would that produce UV light anyway?

Thanks!
Eric

[carolinethorn]

Hi Eric,
Sorry its taken me so long to get back to you.
The things about taking an image of the gel and scanning and quantifying is that you need some kind of stain to measure the DNA bands. The most commonly used stain to visualize DNA is ethidium bromide, which is a carcinogen and i'm not sure what kind of approval you would need to use it. As long as you wear gloves and don't inhale it it should be fine but i don't know what the science fair rules are. Maybe your teacher knows. There are other newer types of stains which are less toxic but more expensive.

I'll try and take a look at ImageJ and see what I can figure out.

As for a heat source, in most incubators the heat source is in a separate compartment and the heat is conveyed by convection. Ordinary lightbulbs don't emit much UV and i guess could be used as a heat source. Might be worth searching old posts on these boards for answers to this Q.

Hope some of this is helpful,
Caroline

[ericjang]

Thanks so much!

Yes, I am planning to use ethidium bromide for my experiment. I considered the alternative stain methods, but figured that it probably is too difficult to obtain SYBER-green or SYBER-safe staining methods. My teacher says ethidium bromide is okay, as long as she handles that chemical when it is being used. As for whether it is okay by the science fair, I will know if SRC approves the experiment.

I have run into a little problem, however. Today, I wanted to test how long yeast can ferment, just to assess what lifespan it has. I noticed that after about 5 hours or so, the yeast is starting to look a little unhealthy. I couldn't find any information on the lifespan of yeast on the internet, as the only results that showed up were about yeast infections and the cell cycle of yeast- none addressed how long yeast could keep dividing. This presents problems, as in my experiment, I run a gel on the same batch of yeast every two days to find telomere length. I may have to change my procedure or my model organism if yeast proves to be too short-lived.

Thanks for looking into the ImageJ software!

Eric

[carolinethorn]

Hi Eric,
Sorry i didn't see this before.
I have to admit I haven't done much yeast cell culture but have done a lot of bacterial and mammalian cell culture and hope I can help (and i expect some other experts will chime in too!).
What conditions are you using? what kind of media are you growing in and what size flask? how much yeast are you putting in to start the culture? and how do they look unhealthy?
-Caroline

[ericjang]

Hi Caroline

Actually, I have some good news. I couldn't find any information on their lifespan in my research, so I conducted a mini-experiment myself. Luckily, yeast is an exempt organism. Over the past two weeks, I ran a mini experiment with one culture of yeast suspended in a sugar solution. I added some sugar every day and warmed up the water in frequent intervals. I have determined that the yeast CAN live for the course of my experimentation cycle, which is at least 10 days.
Also, I figured how to use ImageJ to use densitometry and find the relative area of the bands, measured in pixels or however I would like it. I think the outline of instructions is here: http://www.lukemiller.org/journal/2007/ ... thout.html
I ran some tests with some sample gel images that I found on the internet, and it actually works pretty well.

Now all that remains is the problem of determining whether the telomeres are indeed the largest digested fragments after I run them through with the hindIII enzyme (its probably the only one I am able to get).

[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