Science Buddies: "Ask an Expert"

To whom may concern,

This topic is a branch off of my "Telomere Science Project" topic. I will be discussing all my problems with proper yeast DNA extraction that is a critical aspect of my experiment.

I have spent a good amount of time doing research on how to properly extract DNA for a HindIII restriction enzyme digest. So far, I have mostly found very complicated procedures that involve reagents/equipment I am not in possession of. Since I am unable to do my experiment in a laboratory, I must make do with more "primitive" protocols. Thus, I have spent the last month and a half trying to modify my procedure to accommodate simple tools but yet produce a usable quality of DNA.

I have the following materials to work with:

Distilled Water
Detergent
Salt
Meat Tenderizer
95% rubbing alcohol *(I don't have it on me right now as it is at school, but it's somewhere around that number)
Very small table top centrifuge, capable of around 2000-5000xg (typical laboratory centrifuges use somewhere around 13000 g for separating proteins from DNA).

Here are the main links I have been using to modify my experiment.

http://www.accessexcellence.org/AE/ATG/ ... yeast1.php
This is a very basic yeast DNA spooling procedure for the middle school-high school classroom. I possess all of the equipment necessary for this process, but as an expert has told me, a blender may be impractical for a restriction enzyme digest because the blades are likely to shear the long strands of DNA apart. However, as yeast is notorious for extremely tough cell walls, I need a way to lyse the cell walls without cutting the DNA too much or using expensive materials, such as a vortex mixer with glass beads.

https://www.funakoshi.co.jp/data/datash ... /78870.pdf
This procedure describes a non-conventional procedure using strange reagents to isolate the DNA. This process is more suited towards a professional laboratory, but the principles generally remain the same. I believe that as for the TE buffer used to re-suspend the DNA, distilled water can be used instead.

http://userpages.umbc.edu/~jwolf/m1.htm
This link describes a bacterial DNA extraction, but the second half of the process seems more oriented towards what I am doing. As for the washing procedure mentioned in this link, this step seems to be extremely important for my experiment because I plan to digest the DNA for later agarose gel electrophoresis. It is also mentioned in the second link as one of the final processes. As mentioned before, I have in my possession 95%* isopropyl for the DNA washing process. Unfortunately, upon reading this website thread
http://www.protocol-online.org/biology- ... 30158.html
I am concerned about the overall isopropyl alcohol concentrations for my experiment. Do I need precise concentrations of ethanol for different parts of the experiment? If so, is it okay to simply dilute a 95% concentration into a 75% concentration with water? The link mentions something about how ethanol has a natural affinity for water over DNA, but I am not really sure what that means.

Finally, I am not sure how to "wash" DNA. The way I see it, I just add 70-75% alcohol to the spooled DNA, and then spin it down in a centrifuge.


Finally, here is the restriction enzyme digest and agarose gel electrophoresis protocol I will be using
http://www.methodbook.net/dna/restrdig.html
http://www.methodbook.net/dna/agarogel.html

I spent last week trying DNA extractions, all of which failed quite miserably. This is my old procedure, which yields very faint results on an agarose gel.
1.) Scrape off some yeast cells from each petri dish and suspend in 2.5 ml of distilled water in a test tube.
2.) For each of the 12 batches, Add 2.5 ml detergent/water solution.
4.) Add 1 ml of meat tenderizer/water solution, and invert solution again. Add 1g of salt and continue mixing.
6.) Place 7.5 ml of mixture into a microfuge tube.
7.) Spin the mixture in the microfuge for 2 minutes
8.) Transfer the lighter layer of DNA into a new tube, and discard the protein precipitate.
9.) Pour about 7.5 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.
10.) Spin the tube in the microfuge for 2 minutes so that the DNA precipitates at the bottom of the tube.
11.) Siphon off the excess liquid from the tube.

I have been notified that at this level, the centrifuge I have is not powerful enough to sufficiently separate the proteins from the DNA, let alone get the cells to break open in the first place.

As a result, my main problems, in a nutshell are:
-getting the cells to break open so they release their cell contents
-Finding out how to separate proteins from DNA- should I spool DNA and then separate proteins, or should I separate proteins and then spool DNA?
-figuring out the issue with isopropyl concentrations and how they are related to DNA solubility.


Sorry for my extremely long and verbose post- this thread is more like a summary of my DNA extraction problems taken from my other topics. I hope this won't be too confusing!

Sincerely,
Eric Jang

Dear eric,

I think that you should really look into contacting a nearby college, laboratory, or hospital with laboratories that have equipment, protocols, and professionals that you can work with. I have personally performed the techniques you have described and definetly think that it would be beneficial to work with a professional. Especially for the gel electrophoresis, I don't think that you have the necessary equipment or chemicals for this procedure. The equipment and chemicals for this are dangerous (ex. ethidium bromide and uv light) and are unable to be purchased by anyone except a lab. Remember to take precautions while working with different chemicals and yeast. My advice to you is to look up different colleges, labs, or hospitals and email scientists who may be able to help you.

good luck,
scienceexpert123

Hi ScienceExpert123,

Thank you for your suggestion! Yes, a laboratory environment always has the best materials for experiments, as they have the most accurate, sterile, efficient, etc. protocols and equipment. Since the deadline for the science fair is fast approaching, I doubt that I will be able to get a connection with a lab that is willing to sponsor my project. I have already sent numerous mentorship requests in the past that were all turned down.
As a result, the SRC has allowed me to conduct my experiment at my high school, where I have access to gel electrophoresis equipment, a UV lightbox, and some EtBr. I have been able to improvise some pieces of lab equipment, such as my own petri dish incubator and imaging system.
Of course, the materials I currently have are definitely not 100% accurate, as I lack the proper primers for a PCR experiment or hybridization probes for a southern blot. Thus, I have to make do with the things I have.

Thank you for your concern!

Sincerely,
Eric Jang

Hi,

I have been able to answer some of the DNA washing techniques own my own, but I still have a few more questions regarding this issue. Basically, the DNA washing procedure consists of adding 70% ethanol diluted in water to a DNA pellet. The ethanol concentration is high enough to still precipitate the DNA (like a spooling experiment), but low enough to dissolve the extra salts. That way, when the tube is mixed and then centrifuged, you can easily drain the supernatant and obtain a purified DNA sample.

I have been mostly able to derive information from this site
http://www.protocol-online.org/biology- ... /5564.html
however, I am still unsure as to whether I can substitute the ethanol as mentioned in the experiment with isopropyl alcohol. If any one has any insight for any of my other problems, I would greatly appreciate your help. As I mentioned earlier, I am having trouble with a simple but effective DNA extraction protocol with the materials I have on top of lysing the cells easily.

Thanks!
Eric Jang

dear eric,

I advise you to do a google search of "genomic dna purification protocols". You should get some good protocols for your experiments. Again, I would like to encourage you to seek professional (contact a college, lab, or hospital with labs) help because I really think that you will get better results, will be safer, and you can learn a lot more. Referring to using isopropyl alcohol, from my experience, you need to use both ethanol and isopropyl alcohol to wash the dna. Also, you probably need buffers for this experiment, which you may not be able to purchase by yourself, so again I would advise you to contact a professional.

good luck,
scienceexpert123

Hi ScienceExpert123,

Thank you for your suggestion! The link was very helpful for double-checking my DNA protocols.
Regarding your suggestion to seek professional help, I completely agree with you; laboratories/hospitals/etc. have undoubtedly the most accurate/safe/enriching methods of use for a experiment protocol such as mine. I would gladly accept any opportunity to work in a lab, but as the science fair deadline is fast approaching, I feel that I no longer have the time to count on mentorship from a lab. I have sent tens of mentorship requests in the past, but all of them have been turned down (in fact, all except for a few did not even receive replies). Thus I can only resort to my current protocols and experiment environment. If you know anybody that will be willing to mentor my project immediately, I would greatly appreciate any connections to a lab I can get.

In the meantime, I have managed to solve most of my experiment problems for the time being save for one. I posted this problem on a separate thread, but as no one has been replying to my post on that topic, I will post my issue here in hopes of reaching more help. I have copied my problem below from the aforementioned topic.


I am a little bit confused with the reagents I must combine for the HindIII restriction digest in my experiment:

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?

Sorry if this causes any confusion here,

Thank you so much!
Eric Jang

Hi Eric,

Glad to see you are still persisting despite the obstacles. I hope your resulting presentation will be successful and also help you get a mentor for next year (its worth talking to the science fair organizers to see if they know anyone that can help you, you might have to work on something not directly related to your favorite topic but it would be worth it to get the experience).

Back to the question in hand : 5x buffer and diluting.
Yes the buffer needs to be diluted but it can be diluted by both the water, the DNA solution and the enzyme so you look at its proportion in the final volume.
In the example on the link you posted they had 10x buffer. If you add up the total the final volume is 10microlitres and the 10x buffer was 1 microlitre so it was diluted 1 in 10.
You have 5x buffer so if you used the same recipe on the link it would be
* 2µl 5x Buffer
* 5.5µl H2O
* 2µl DNA
* 0.5µl Enzyme
The final volume is again 10microlitres and the 5x buffer was diluted 2 in 10 or 1 in 5, to make space for the extra buffer you use less water.

Best of luck,
Caroline