Steve Jobs, 2005 Stanford Commencement Address

Remembering that I’ll be dead soon is the most important tool I’ve ever encountered to help me make the big choices in life. Because almost everything — all external expectations, all pride, all fear of embarrassment or failure – these things just fall away in the face of death, leaving only what is truly important. Remembering that you are going to die is the best way I know to avoid the trap of thinking you have something to lose. You are already naked. There is no reason not to follow your heart.

We who work to combat the diseases of old age that lead to decrepitude use the same realization to spur us forward in the fight to save lives.  Is there any other rational course of action?

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Thanks to the European Genetics and Anthropology Blog for this DIY Genetics experiment:

If you bought a genome-wide scan at 23andme and/or deCODEme, then you have access to your raw data, which gives you the option of going beyond the bio-geographic analyses offered by these companies. For example, you can use various programs to compare yourself to publicly available samples from around the world. Structure is one of the more popular tools for this sort of thing, so here's a guide how to set up a quick analysis using Structure and a data sheet from Kosoy et al. 2009:

- Download the 2.3.2 Beta version of Structure, with the graphical front end, which makes things a lot easier.

- Extract the following 125 SNPs from your raw data. Actually, 128 are listed on that sheet, but only 125 currently available at 23andme, although that's not a problem.

- Convert the genotypes to integers, as per the instruction sheet above. For example, if you're AG for rs731257, then convert that to 12 (ie. A=1, G=2). The three missing SNPs, as well as any no-calls, should be listed as 55.

- Download the sample data sheet, and add yourself to it. Make sure you look exactly like all the other samples on there, so you'll need to add the various tags that precede the genotypes. For example, instead of "EURA CEU CEPH1334.10 1", try something like "EURA POL Myself 1", or if you're African American then maybe "AFR AME Myself 2".

- Start Structure and load up the data sheet by going to "File" and then "New Project". Fill in the necessary fields in the Project Wizard, such as: Number of individuals 639; Ploidy of data 1; Number of loci 128; Missing data value 55. Then tick the following boxes: "Row of marker names", "Data file stores data for individuals in a single line", "Individual ID for each individual", "Putative population origin for each individual", "USEOFPOPINFO selection flag", and finally "Sampling location information".

- Define the parameter set (ie. go to "Parameter Set" and then "New"). The length of burn-in period should be at least 10,000 and the number of MCM reps about 50,000. Of course, to save time you can reduce both, especially if you're not too worried about a bit of noise. On the other hand, if you want to minimize noise as much as possible, then go up to something like 100,000 burn-ins and 500,000 reps. But be warned, runs like this can take days.

- Press the "!" button, specify the number of clusters (K) you'd like to divide the samples into, and click "OK". Alternatively, you can let the program work its way from K2 to whatever; Project > Start a Job > pick the parameter set > specify the K range (for example, from K2 to K6) > press the "Start" button.

Here are my results at K4. Obviously, if you're of overwhelmingly European origin, it's unlikely you'll get anything below 99% European/West Eurasian with these 125 markers. Much larger sets of SNPs are needed to get more detailed admixture estimates, and to break down the intra-West Eurasian and intra-European components.

Indeed, if you're good with Excel and Access then it's even possible to go up to something like 500,000 SNPs. HapMap and HGDP samples are available online, although the latter are presented in a somewhat different way than the 23andme raw data, which is a real pain because it takes a lot of work to overcome. Also, there are other settings you can try to see how they affect the results, like turning on LOCPRIOR, which tells Structure the putative origins of the samples. You can use different data formats too, examples of which are shown on the Structure home page.

Roman Kosoy et al., Ancestry informative marker sets for determining continental origin and admixture proportions in common populations in America, Human Mutation 2009,
Volume 30 Issue 1, Pages 69 – 78, doi: 10.1002/humu.20822

Hubisz M. J., Falush D., Stephens M., Pritchard J. K., Inferring weak population structure with the assistance of sample group information, Molecular Ecology Resources 2009. DOI: 10.1111/j.1755-0998.2009.02591.x
http://eurogenes.blogspot.com/2009/11/running-your-own-structure-analysis.html

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By Guy Montague-Jones, 17-Nov-2009

Related topics: Research, Minerals, Cardiovascular health

Taking selenium supplements may increase cholesterol levels by as much as 10 per cent, according to a new study.

http://www.nutraingredients.com/Research/Selenium-supplements-linked-to-high-cholesterol/?c=0dQornGJR3qf6dGwol2Fng%3D%3D&utm_source=Newsletter_Subject&utm_medium=email&utm_campaign=Newsletter%2BSubject

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By Stephen Daniells, 10-Nov-2009

Related topics: Research, Minerals

Formulating iron-enriched fruit juices with milk may improve uptake of the mineral, suggests new research that offers a way of boosting iron intake for people at risk of deficiency.

http://www.nutraingredients.com/Research/Milk-may-boost-iron-uptake-from-fruit-juices/?c=0dQornGJR3q46in86HBIPg%3D%3D&utm_source=Newsletter_Subject&utm_medium=email&utm_campaign=Newsletter%2BSubject

Posted via email from healthystealthy health hacks

By Stephen Daniells, 09-Nov-2009

Related topics: Research, Antioxidants, carotenoids, Phytochemicals, plant extracts, Cancer risk reduction, Cardiovascular health, Cognitive and mental function

Nano-sized curcumin capsules may boost the body’s uptake of the ingredient, and enhance its potential to prevent colon cancer and Alzheimer’s disease, suggests a new study from Japan.

http://www.nutraingredients.com/Research/Nano-curcumin-could-boost-spice-s-health-benefits/?c=0dQornGJR3r%2FT4mfAXAtFA%3D%3D&utm_source=Newsletter_Subject&utm_medium=email&utm_campaign=Newsletter%2BSubject

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THE MACGYVER PROJECT: GENOMIC DNA EXTRACTION AND GEL ELECTROPHORESIS EXPERIMENTS USING EVERYDAY MATERIALS

Abstract:
DNA extraction and separation by agarose gel electrophoresis is a simple and exciting process that anyone can perform. However, the high cost of specialized equipment and chemicals often hinder such an experiment from being carried by members of the high school community. Here, we describe a cost effective way of extracting and electrophoresing DNA under a prescribed MacGyver limitation – that is using only materials available from a grocery store or shopping mall. * * *

In order to carry out this project, we decided to first divide the procedure into three specific sections, each to be addressed individually. Doing this, you find that the following challenges are present. They are: (i) extraction of DNA, (ii) gel electrophoresis of DNA and (iii) visualization of DNA.

Extraction of DNA in a Research Setting:
In a conventional research setting, the first step in extracting DNA involves breaking open the cell’s membrane by using physical or chemical means. Examples include the use of sonication (sonic waves), homogenization equipment (blender, french press, mortar and pestle) or selective detergent use. To us, exploration of detergents (or soaps) or physical steps (mortar and pestle) were the most obvious choices.

Once the cell/tissue has been lysed, usually subsequent steps involve an attempt to purify or enrich the sample for your DNA (i.e. get rid of the other stuff). This can be done in a number of ways, many of which are not technically feasible under our MacGyver rubric. However, a cell lysate can be easily enriched for DNA using alcohol to selectively precipitate DNA, forming it to clump into a “snot” like entity. Consequently, we have decided to focus on the extraction of genomic DNA since this type of sample works best under the alcohol precipation protocol and is also the most abundant DNA species for ease of visualization at both the extraction and gel steps.

A genomic prep is also interesting from an educational point of view. A teacher could pose ‘why do people want to obtain genomic DNA?’ – a question which can lead to numerous discussions pertaining to the cloning of new genes, organisms (Dolly the Sheep); having a source of DNA for fingerprinting purposes (CSI, forensics), or even the preparation of a sample for the sequencing of the organism’s entire genome (Human Genome Project). Focusing on genomic DNA also allows a student to explore samples from different sources, where there is a reasonable possibility that the differences in genome size can be discerned. As a bonus, the precipitation of DNA into a ‘snot’ like form adds an added ‘wow’ factor to the activity.

MacGyver Extraction of DNA:
Reagents and materials for this portion of the experiment are fairly straightforward. Here, one needs to find a tissue source, some manner of physical breaking, clean water/buffer solution, a soap, and an alcohol. In addition, some type of container to do this in will be required, preferably one that is transparent in nature.

Tissue Source:
Although the chemical characteristics of the DNA isolated is practically identical from organism to organism, the cell in which it is housed can vary greatly. Consequently, choice of tissue is the largest variable, but arguably the best element for a student to play around with. We have tried extracting DNA from onion, split peas, corn, yeast, bean sprouts, wheat germ, kiwi, banana and even human cheek cells, all with varying degrees of success. We find that overall, good DNA can be achieved with any fresh produce or grain material, as long as the experimenter is willing to play around to find optimal conditions. Overall, however, we recommend onion or banana tissue as the best sources of low maintenance DNA extraction. We also recommend wheat germ, in that a “fast” procedure exists that works very well. Finally, DNA isolated from a student’s own cheek cells is an interesting alternative, since it brings in a personal element to the activity. However, it should be noted that isolation from cheek cells is less reliable.

Physical Step:
Depending on the tissue you use, a physical step may be warranted. This could be as involved as cutting up the tissue and then using a mortar and pestle, or as minor as banging around with a wooden chopstick. In general, plant material and meat cuts would benefit from such a step, since plants have a tough cell wall to contend with, and meat usually contains tough muscle striations.

Buffer Solution:
This is the fluid that the sample needs to be immersed in, and as such has the role of simply keeping your DNA safe. There are many possible MacGyver type buffers that one can use for the extraction procedure, which all basically work (see Table 1). We should note, however, that distilled or bottled water should be used. Tap water appears to have a minor effect on the extraction procedure, and is actually very detrimental in the subsequent gel steps described later.

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TABLE 1: BUFFER RECIPES*

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A: “Saline Buffer”
0.9% saline (contact lens solution)

B: “Regular Buffer”
1.5g table salt (NaCl)
5.0g baking soda (sodium bicarbonate)
to a final volume of 120ml with water

C: “Acidic Buffer”
1.5g table salt (NaCl)
5.0g baking soda (sodium bicarbonate)
5mL of vinegar
to a final volume of 120ml with water

D: “Proteinase Buffer”
1.5g table salt
5.0g baking soda
2.5mL Complete eye solution or 2 protein tablets (Complete brand)
to a final volume of 120ml with water

E: “Meat Tenderizer Buffer”
100mL hot water
3g meat tenderizer

*Note that water alone can also be used effectively.

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Soap/Detergent:
Liquid dish washing soap was generally used. In terms of specifics, we found better results when the soap was colourless, and found that unscented versions may work best overall. Essentially, most soaps available at grocery stores have additives that play roles in preservation, scent, colour, stain removal etc, etc. Although these things are usually not an issue with the technique, choosing the simplest soap is a good thing to troubleshoot with if you are not having success with your extraction. In general, we found that using about 5ml (or less) of detergent per 120ml solution works well.

Filtering:
Depending on your tissue choice and also the amount of tissue you have decided to use, it may be pertinent to include a filtering step to remove the debris. This allows a better visualization of the DNA snot effect at the end. Filtering can be easily accomplished using coffee filters, or cheese cloth. Note that when using cheese cloth, you will need use 3-4 layers of cheesecloth. Coffee filters are generally easier because they are cheaper, more accessible, and easier to cleanup.

Precipitation with Alcohol:
Precipitation of DNA is done by contact with alcohol. This alcohol is available at pharmacies as rubbing alcohol of the 99% iso-propanol variety. Alternatively, most schools will have access to 95% ethanol stocks. The alcohol should be added in a layering manner so that one can see the DNA forming at the interface between your sample and the alcohol. You should add at least two times the volume of your original sample. If you do not see “snot” forming, then mixing everything together should get the desired effect. Note that if you intend to resort to mixing, then the filtering step may be especially necessary for ease of visualization.

DNA precipitates under alcohol treatment because it is naturally hydrophobic and as such will tend to clump together if the solvent is not optimal (i.e. water). As a result, adding an alcohol to your prep will mean that DNA is not completely solvated in its optimal environment (water), and will therefore aggregate, and precipitate out of solution. It should be stressed that the degree and ease of clumping will depend on the amount of DNA present, and also the concentration at which it exists. Therefore, if you see minimal amounts of DNA, you should be able to correct this by (i) trying to isolate from more tissue, and (ii) resuspend the material in a smaller volume of fluid.

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TABLE 2: SUGGESTED MACGYVER GENOMIC DNA EXTRACTION PROTOCOLS:

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GENERAL PROTOCOL
1. If necessary, slice up DNA source of choice. Use an amount about the size of a strawberry.
2. Using a mortar and pestle, grind up sample while gradually adding 10mL of prepared buffer solution with the detergent already added. Grind for at least 5 minutes with all of your weight and strength to ensure that you break open the cell membrane and reach a creamy soup consistency. If the sample is too thick after grinding, add more saline solution to achieve the optimal thickness so that the liquid portion of the sample is able to pass through the filter, while the larger cellular material remains behind on the filter. Note: If the DNA sample is frozen, it is considerably easier to grind.
3. Filter your sample’s juice into a small beaker. Let the solution drip into the beaker until all of the liquid has passed through the filter. If this takes too long, simply squeeze all of the juice from the sample through the filter.
4. Add 2 volumes (this means approximately two times the volume of the sample present) of ice cold alcohol down the side of the beaker with a straw or pasteur pipette. Do this step slowly to enable the alcohol to form a layer on top of the juice layer. As you let the beaker sit, the DNA should precipitate. The longer you wait, the more DNA you should see. If you don’t see precipitation, gently mix everything together.
5. The DNA precipitates should resemble a thread of translucent white snot,at the interface between the juice and alcohol. After a considerable amount of time, the DNA may eventually float to the top of the alcohol layer.
6. You can remove the DNA with a wooden popsicle stick or glass rod. DNA adheres well to the wood.

QUICK PROTOCOL
1. Place a teaspoon or less of wheat germ in your cup.
2. Add about 10ml distilled water and crush gently with a popsicle stick/chopstick for 1 minute.
3. Then add a squirt of dish detergent and crush gently with popsicle stick for 2 minutes.
4. Slowly pour alcohol down the length of the stir stick, layering the alcohol on top of the water.
5. Set the container down on a table and look for the DNA at the interface between the alcohol and water.

CHEEK CELL PROTOCOL
1. Measure 10 ml of “regular buffer (from Table 1), pour buffer in mouth and swirl around cheeks for about 1 minute.
2. Spit the water back into a container, preferably something relatively narrow like a test tube.
3. Squirt a bit of liquid soap to the sample and mix well with popsicle stick. If you can mix by inversion, then do so gently about 20 times.
4. Add 10 ml cold alcohol slowly to the sample and make sure to pour it at an angle down the side of the test tube so that two layers are formed. Do this very gently, with a straw, etc. It’s important that the two layers are not disturbed.
5. Wait for about 10 minutes and the DNA will appear afloat on the alcohol layer.

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Gel Electrophoresis of DNA in a Research Setting:
Electrophoresis is a way of separating molecules based on charge and size. In our case, we want to separate different sizes of genomic DNA molecules obtained from fruits, vegetables and yeast. Generally, polysaccharide polymers such as agarose or acrylamide are used to form the electrophoresis gels. Because DNA is negatively charged, one can force it to travel through the gel by applying an electric field in the system. Normally, this is achieved by using special gel apparatus designed to facilitate the production or casting of the “gel” as well as allow a platform to immerse the gel in an ion containing buffer to create an electric field. Such an apparatus will run a minimum of about $500, and power packs normally used to deliver ~80V of voltage can run in a similar price range. Consequently, this aspect of the MacGyver project is arguably focused on cost savings and concentrates primarily on finding a substitute for agarose and directions for producing a gel apparatus.

MacGyver Gel Electrophoresis of DNA:
Gel Material:
Agarose is a component of seaweed and as such is a refined purified molecule derived from a common food thickener known as “Agar Agar.” which incidentally can be easily found at most oriental style food stores. “Agar Agar” can be purchased in either a flake, noodle or powder form. Try to ensure that it does not contain any additional ingredients (such as glucose) as these ingredients may interfere with the formation of the gel matrix. To make the gel, it is recommended to use “Agar Agar” in the powder form rather than the flake or noodle form. The other larger forms tend to require cutting and additional filtering which is problematic and very messy.

Running Buffer:
You will need to prepare a running buffer which is required to make the gel, and also required as the fluid that will ultimately immerse your solidified gel to allow the electric field to be conducted. The Macgyver running buffer recipe is as follows:

- 0.05g of NaCl (this is the principle ion)
– 2g of Baking Soda (Sodium Bicarbonate)
- bring to 1L with distilled bottle water

pH’d using pet store aquarium pH kit to approximately pH7.5. (we used alkaline buffer made by Seqchem).

Gel Apparatus:
Although a research grade gel box is costly, it is in fact a relatively simple piece of equipment. In essence it is a large container (let’s call it a buffer chamber) that can hold fluid, whereby opposite ends are connected to a power source setting up a positive/negative electrode scenario. The buffer chamber needs to be able to conveniently hold a smaller container (let’s call this the gel casting chamber) that is used to make and hold the gel. Furthermore, the smaller gel casting chamber needs to fit in such a manner as to be in the middle between the two electrodes of the buffer chamber.

Assembling this electrophoresis box should be quite straightforward and even enjoyable for those that like to “make things.” We have included here a cartoon guide (Figure 1) for making these two chambers out of common plasticware (tupperware, soap dish, etc). For the electrode connections, we were limited to stainless steel screws (5cm) and stainless steel wire (20 gauge), which worked fine. However, a more elaborate electrode system would be easy to make with a visit to a proper hardware store.

As an alternative to using tupperware, we have also found Lego to be extremely useful in custom making a buffer chamber to fit your gel casting chamber. Note that Lego chambers will need to be lined with a water tight material, but recently Glad has developed a new “Glad Press’N Seal” material which works perfectly and is easy to handle.

Finally, a “comb: will need to be made. This is a contraption that allows small wells to be formed in your gel. Here, we found lego to be especially useful, but in a bind, we also made combs by cutting out pieces of plastic, or taping the teeth of a real hair comb.

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FIGURE 1: MACGYVER GEL BOX CONSTRUCTION NOTES.

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Gel Preparation:
Using the powdered “Agar Agar” you will want to make a 1.2% to 1.5% gel (w/v or 1.2g to 1.5g per 100ml of running buffer). In a separate container, (a flask for instance) weigh out the required amount of “Agar Agar” and add the required amount of running buffer (specific amounts will be dictated by the size of your gel casting set-up). The flask should be large enough so that the “Agar Agar” solution forms a 2 cm layer at the base. This is required to ensure a large surface area in contact with the hotplate for effective melting. It is recommended to use a hot plate and continued stirring to heat the agar solution evenly. An alternative is to use a microwave, but with this method one must use a low heat level at short intervals while swirling the mixture frequently. If this procedure is not carried out carefully, the solution will likely overboil and create a large mess. Note that if the “Agar Agar” solution is not completely dissolved, this will greatly affect the mobility of the DNA as it travels through the gel. Furthermore, undissolved bits of agar will be stained during the staining procedure, making it difficult to view the bands of DNA.

Note: some schools have access to the “Agar” used to make bacterial plates. This material works very well in pouring gels (much better than “Agar Agar”) We recommend making a 1% w/v gel for genomic visualization purposes.

Make sure that you have sealed the open ends of your casting gel chamber with masking tape or similar material. Pour the melted mixture into your casting gel chamber. Remember to also insert the “comb” so that well formation can occur. It will take approximately 20 to 30 minutes at room temperature for the gel to solidify. During this time do not disturb the gel. The gel is quite delicate, so be very careful when you remove the comb before loading your sample

The gel should be poured to about a 0.5cm to 1.0cm thickness. Thicker gels allow the opportunity to make deeper wells, thereby allowing a larger sample to be loaded. Thinner gels should run faster. Note that if the gel is too thin, it may float when immersed. Try to use the gel as soon as possible. Although you can wrap it in plastic wrap and store it in the fridge overnight, they will work better when used fresh.

DNA Sample Preparation:
Your genomic snot DNA can be removed at the end of the extraction procedure using a wooden stir stick. The DNA should then be dipped in a 70% alcohol solution which facilitates removal of excess salts. This is very important for effective dissolving as well as effective gel running. The DNA can then be air dried for approximately 10 minutes to evaporate residual alcohol, and then dislodged into a small volume of running buffer (the smaller the better, i.e. <0.5mls). One should note that genomic DNA can often take days to dissolve properly. We suggest allowing the DNA to dissolve overnight at room temperature (if you have access to temperature up to 50C, then that is best). Be gentle to your sample as the large genomic DNA is very prone to shearing. Once the overnight dissolving step is finished, consider this your DNA sample regardless of whether it has dissolved to completion.

Your sample will then need to be treated with a loading buffer. This is essentially something that will cause your sample to be viscous so that it can indeed “sink” into your wells during loading. Often, a loading buffer also has a dye, which will travel in the same direction as the DNA, and also makes the gel loading easier to see.

Our recipe is as follows:
– 0.5ml glycerol/glycerine (available at the pharmacy section)
– 0.1ml distilled water
– several drops of Club House Red Food Colouring (note that choice of dye can affect outcome greatly – we didn’t have a lot of success finding something good here, so a last resort would be to use without a dye).

This loading buffer can be used as 5X to 10X meaning that for 0.5ml of sample, you would need to add 0.05 to 0.1ml of loading buffer. Mix carefully. Your sample is now ready for the gel.

Gel Running.
Place your solidified gel (in gel casting chamber) within the larger buffer chamber. Add running buffer until the gel is immersed such that there is at least 3mm of fluid above the gel. Keep in mind that the more fluid you add, the slower the gel will run.
To your wells, load as much sample (with loading buffer) as possible. This is probably best done with a plastic stir stick attached to some type of rubber bulb. Essentially you would like to assemble something that can deliver fluid through a small tube. Once all the samples have been loaded, you will want to connect the electrodes to a series of 9V batteries. Your DNA is negatively charged so you want to position the positive electrode at the end “away” from the wells. Basically, one battery will suffice but will be very very slow (overnight run scenario). 5 to 7 or them lined up in a series circuit should deliver a good amount of voltage. Note that when the gel is running, DO NOT stick your finger in the fluid. Depending on the number of batteries you use, as much as 100mAmps of current is delivered (enough to give you a small shock).

When the circuit is running, a good visual check is to see that bubbles are forming from the wire electrodes, and usually most visible at the positive end.

The optimal amount of time to run the gel is, frankly, something that is difficult to predict, as it depends on the size of your gel, the thickness of your gel, the amount of running buffer in the system, the amount of voltage applied, and even the wiring set-up used. Consequently, this is the one thing where you will definitely have to play around. However, with a 5 to 7 battery set-up, you will need at least a minimum of 1hour, and possibly more. In addition, if differentiating genomic preps is in order (i.e. different genome size), you may need to experiment further with run time to see this potential difference.

Visualizing DNA in a Research Setting:
Upon completion of electrophoresis, the location of the bands need to be visualized. A common way to detect the bands is to stain the gel with ethidium bromide, which fluoresces under ultraviolet light to view the DNA. Unfortunately, both ethidium bromide and ultraviolet light are hazardous (Ethidium is higly carcinogenic and UV light can burn a person’s eyes without proper safety measures) and are therefore, not ideal for use among high school students.

MacGyver Way to Visualize the DNA:
To visualize the DNA bands, the gel was stained in a Methylene Blue solution. Methylene Blue consists of the salt methylene blue chloride. In water, the salt disassociates into a positively charged methylene blue ion that is colored blue and a negatively charged chloride ion, which is colorless. This blue chromophore is then able to bind to the positively charged DNA in the gel. Methylene blue is a convenient stain to use in the lab because it is chemically safe, reusable, and detects the presence of more than 20ng DNA/band.

More importantly, methylene blue can conveniently be found at most pet stores. It is generally sold as an aquarium disinfectant at a 5% concentration. The best gel staining results were obtained immersing the finished gel in a 0.02% methylene blue (in distilled water) solution overnight at room temperature. Using this protocol, destaining excess the excess colour with distilled water incubations was not required. We found that higher concentration solutions tended to stain the gel too dark making it difficult to differentiate the background from the DNA bands. This was observed even after destaining with distilled water. Also, if the “Agar Agar” powder was not completely dissolved in the gel, the non-dissolved flakes were stained dark blue, preventing the formation of distinct, visible bands.

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FIGURE 2: CORN GENOMIC DNA PREP MACGYVER STYLE.

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Yes, it’s faint! But it’s there…

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Conclusions:
Overall, we have described an effective outline of methods to perform some basic molecular biology techniques under the MacGyver limitation (Figure 2). We should stress however that doing this in your own classroom will inevitably require some working out of your own. This is due to a multitude of considerations such as “Do I have enough DNA?” “what is my gel set-up” “what type of specialized reagents do I have at my disposal to make things even more efficient?” etc. However, it is hoped that the information presented here will make your troubleshooting as smooth as possible. Good luck!

Sphere: Related Content

http://www.scq.ubc.ca/the-macgyver-project-genomic-dna-extraction-and-gel-electrophoresis-experiments-using-everyday-materials/

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Posted on: Thursday, 12 November 2009, 09:38 CST

Trigonelline, a compound in coffee, has been found to be estrogenic. Though the studies have not been conducted to determine recommended consumption amounts, scientists say the compound, “trig,” may be a factor in estrogen-dependent breast cancer but beneficial against colon cancer development.

“The important thing to get from this is that ‘trig’ has the ability to act like a hormone,” said Dr. Clinton Allred, AgriLife Research nutrition scientist. “So there is a tie to cancer in the sense that we are looking at estrogen-dependent cancer cells. But that doesn’t suggest that it would actually cause the disease. I don’t believe there should be any concern about drinking coffee at this point.”

His report was published in the Journal of Nutrition. Allred’s lab studies dietary compounds that can mimic the hormone estradiol – the primary hormone in women. His main focus has been to look at how estrogen protects against the development of colon cancer. Estradiol is one of three estrogen hormones. “There’s a history of these compounds in crops such as soy,” Allred said. “Soy has a number of different compounds that actually can mimic estradiol in several disease states some of which are good and some of which have the potential to be more deleterious-type effects.”

Allred said a former colleague mentioned an interest in finding the properties of “trig” – a natural compound used in traditional Indian culture for post-menopausal women.

Because the chemical structure of “trig” was so unlike estradiol, Allred didn’t think the compound would be estrogenic.

“Estrogen-dependent tumors in the presence of estradiol will grow faster,” Allred said. “If you use those cells in a laboratory setting, you can determine whether something is estrogenic because they will literally make a tumor grow faster.”

He said that a series of experiences and different approaches showed that “trig,” a vitamin derivative, was fairly estrogenic at very low concentrations.

“We haven’t gotten as far as to suggest that if a woman had the disease that it would necessarily be a problem. But what we’ve proven is that the compound is estrogenic or can be at certain concentrations and doses,” Allred said.

He added that “trig” is in coffee beans, though in different amounts depending on the variety of coffee bean. The two major types of coffee beans used for what is consumed in the U.S. both contain it, he said.

“The more you roast a coffee bean, the less there is,” Allred said. “But the most critical aspect is that when you do a water extract of ground coffee, which is basically how you make a cup of coffee. It does in fact come out in the water, so we know it is in a cup of coffee.”

Nevertheless, the researchers have no idea what the exposure level would be or whether a particularly exposure – say from one cup of coffee – would be in the range seen in the laboratory tests.

“It is way too early to say that drinking a cup of coffee is exposing you to something that is definitely going to be estrogenic. All we know is that there is a compound in there that can be estrogenic in our systems. That is really the take-home message,” Allred said.

Allred also cautioned that people often narrow one compound in a food without considering the total mix of compounds and how they interact with each other or in a human body.

“There is never a single compound when you’re looking at food, and a cup of coffee is a food,” Allred said. “There’s a whole bunch of other things in it. There’s caffeine. There’s actually a little bit of fat. There are all sorts of others things in a cup of coffee that could interact with this.”

The numerous compounds in each food product means there are complex interactions, he explained, which is why nutritionists advise people that the whole food is better than any individual compound.

“That’s why you can’t take supplements to make up for food. You can never take all the things that are in a carrot and replace a carrot. In the end, you need to eat the carrot,” he said. “We’re a long way from understanding what this compound could do in the context of a food.”

He said a concern is that menopausal women seek over-the-counter phytoestrogen compounds to relieve symptoms such as hot flashes. Women want what they believe to be a natural and/or safe mechanism, he said, because hormone replacement therapy has such a negative connotation.

But, Allred said, researchers estimate that from the time an estrogen-dependent breast tumor begins until it is diagnosed in a woman is about 30 years.

“That means there will be a number of women out there who will become menopausal, and begin to take phytoestrogens in supplement form,” he said. “The majority of those come from soy. So our concern was, what if a woman becomes menopausal which means her estrogen levels are going to be low, she has estrogen-dependent breast cancer and doesn’t even know it. And now she’s consuming phytoestrogens.

“Physicians would never recommend you be on hormone replacement therapy if you had estrogen-dependent cancer. From a toxicology standpoint, it would that be a bad thing if you were consuming these phytoestrogens in high enough doses. It could be really dangerous.”

A problem is that people believe that natural or plant-derived compounds are automatically safe which is not necessarily always true, he said. Also, consuming a compound in its pure form as a supplement in high doses may not be healthy.

“If we were getting a hormone from an animal, you wouldn’t see people do that,” he said. “The only difference is that this is a plant-derived compound, so they feel it is safe when that may not be so.”

Yet, Allred added, scientists are finding that at least some of these compounds are doing positive things to prevent colon cancer.

“So there’s going to be places that it’s good – just as we’ve seen with estradiol,” he noted. “There are going to be some disease states that it is quite good for and some disease states that you need to be mindful of.”

Still, the compound’s potential as a weapon against colon cancer has the researchers “pretty excited about that.”

“We’re seeing very interesting information as far as tumor formation and the ability of phytoestrogens to prevent colon cancer formation. So any other new, natural phytoestrogen that we are able to identify and relate to the diet, that would be the model we’d bring it in to,” Allred said of possible future studies on “trig.”

He said a hope would be to develop a drug that could treat colon tissue without getting into the entire body, thus exploiting the compound’s mechanism to protect again cancer formation without producing other estrogenic effects.

“It’s really important for us to come up with strategies that we can have the benefits in the colon without the risks associated with (estrogenic compounds),” Allred said.

—

On the Net:

• Texas A&M AgriLife Communications
• Journal of Nutrition

http://www.redorbit.com/news/health/1784991/coffee_compound_brewing_new_research_in_colon_breast_cancer/index.html?source=r_health

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Roasting is where coffee’s flavor is fulfilled. The green coffee beans are heated in large, rotating drums using temperatures of about 550 F (288 C). The tumbling motion of the drums keeps the beans from burning.

Photo courtesy Kona Coffee/Bay View Farm Coffees Roasters take the raw, green coffee beans and turn them into the aromatic beans you find in the coffee shop.

The beans first turn a yellowish color and smell a little like popcorn. After about 8 minutes, the beans “pop” and double in size. The beans have then reached about 400 F (204 C) and begin to brown as the oils within them start to emerge. This oil is called coffee essence or caffeol. The chemical reaction of the heat and coffee essence is called pyrolysis, and is what produces the flavor and aroma of coffee. A second “pop” occurs about three to five minutes later and signals that the bean is fully roasted.

Photo courtesy CoffeeResearch.org Before and After: Green (left) and roasted coffee beans (right)

Coffee roasting is something of an art. Roastmasters use sound, sight and smell to determine when the beans are roasted to perfection. Timing is everything. Roasting time affects the color and flavor of the final brew, so the length of the roasting period depends on the type of coffee desired (shorter for American brew, longer for espresso).

EXCERPTS and IMAGES: Warren Buffett & Bill Gates – Keeping America Great

These are excerpts from the unofficial transcript of CNBC's town hall interview today (Thursday) with Warren Buffett and Bill Gates from Columbia Business School in New York City, as released by CNBC.

Warren Buffett and Bill Gates: Keeping America Great, a 90-minute CNBC Town Hall Event hosted by Becky Quick, airs tonight (Thursday) on CNBC television at 9p and 12:30a ET.

Buffett on Controlling Systemic Risk

Going forward, it's a very tricky thing to figure out how to present excessive leverage, how to prevent off balance sheet arrangements from getting in trouble, or for just having people at the top of major institutions that run risks that they shouldn't be running. We're wrestling with that right now. There should be more downside to the head of any institution that has to go to the federal government to be saved for reasons of the greater society and so far we've been better at carrots and sticks in rewarding CEOs at the top, but I think some more sticks are called for.

Gates on Capitalism

This country still has the best universities, the best science and we're going to tune our system of capitalism.  The idea that you have a lot of short-term loans, covering long-term needs, the amount of leverage that was there. There are definitely some lessons, but the fundamentals of the system, a marketplace driven system where we invest in education, in a great infrastructure for the long term, that's continued.

Buffett on Goldman Sachs

I had more confidence in both the numbers and the management of Goldman Sachs , than any other major firm on Wall Street at the time (Berkshire made its $5 billion investment in Goldman last fall.)  Now, there could've been things happen that would've made Goldman Sachs be next in line. (Goldman CEO) Lloyd Blankfein has said, 'We're 30 seconds behind Morgan Stanley.' This is covered very well in a book by Andrew Ross Sorkin, but I did not think the system was going to go under. I felt Washington in the end would do the right things and I felt if they did the right thing. Goldman Sachs,  I thought it was the best-run operation. I thought its figures were the most solid and I thought they would prosper the most in the future ahead

Gates on Investing in the Future

Industries do have different paces of innovation and so does the IT industry, driven by the magic of software, the magic of the optic fiber, magic of the chip, which doubles in power every couple years.  It's been the industry that not only has been the most exciting, it's also changed the rules for many other industries. You know the idea of information being available, what the online world is like.  That's incredible.  I'd say there are a few other industries that will compete for being exciting for the decades ahead.  The energy business, some approach will provide cheaper energy that's environmentally friendly and there's a lot of science, a lot of business, it's a global thing.  They'll be some great careers there.  Medicine, you know, we haven't solved Parkinson's or Alzheimer's or about 20 diseases of these poor countries and yet, we can be sure that we're on track to do that.  And so those 3 industries, I think, you would do great in.

Buffett on Berkshire's Acquisition of Burlington Northern

We start out with a premise and I can't think of a more sound premise, that there will be more people in this country 10, 20, 30 years from now. They'll be moving more and more goods back and forth to each other and you have the most environmentally friendly and the most cost efficient way of doing that on the railroads. The Burlington Northern last year moved, on average, it moved a ton of freight 470 miles on 1 gallon of diesel. You know that is far, far more efficient than what takes place over the highways.  You have a situation where overall they use a third less fuel, they put far less pollutants into the atmosphere than trucks will.  One train will supplant 280 trucks or so on the road.

So, the rails are in tune with the future and there's, I like the West, I like the 30-some thousand miles that Burlington has and if this country has a poor future, rails have a poor future and I'm willing to bet a lot of money that, 34 billion to be specific, that 10 years from now, 20 years from now, 50 years from now, there'll be more and more moved by rail and it will be better for the country and it will be better for the shareholders at Burlington Northern.

Gates on Google

They're hiring a lot of smart people. They got into the lead position in search, which is incredibly profitable, to be number one in that. They may get a little competition as time goes forward, but they're a great example of what can happen. You know, 2 young guys who got together, pursued an idea and created a success that's absolutely gigantic and we all, uh, hopefully use search engines, hopefully a variety, and we benefit from that.

Google:

Buffett on Gates

What I really most admire about Bill is the view he has about what he should do with the wealth he's accumulated. I mean, as he said, he was very lucky, he was born in the right country at the right time, with the right wiring and all of that sort of thing.  But in the end he knows he's the beneficiary of a terrific society and not everybody gets the long straws, like he and I did, so he is, and he has this view that every human life worldwide is the equivalent of every other human life and he's backing it up, not only with money but he's backing it up with his time. And his wife Melinda is backing it up with her time, and they are really going to spend the last half of their lives or so using both money, talent, energy, imagination, all to improving the lives of six and a half billion people around the world and that's what I admire the most.

Warren Buffett and Bill Gates: Keeping America Great, a 90-minute CNBC Town Hall Event hosted by Becky Quick, airs tonight (Thursday) on CNBC television at 9p and 12:30a ET.

http://www.cnbc.com/id/33898438/site/14081545?__source=yahoo%7Cheadline%7Cquote%7Ctext%7C&par=yahoo

Posted via email from healthystealthy health hacks

Intro: 5 minute DNA Extraction in a Shot Glass

Despite its exotic-sounding name, DNA is ubiquitous – it can be found in every cell of every living thing and almost everywhere on the planet. Nonetheless, we rarely come face-to-face with the molecule itself – and it’s not because DNA is difficult to find or isolate! In this instructable, we’ll show you how to isolate your own DNA with little more than some dish soap, table salt, high-proof alcohol, a shot glass, and a bit of your own saliva.

It only takes a couple of minutes, and after you’ve isolated your own DNA, you can either drink it back down in a tasty “DNA shot” (great party trick) or better yet, purify it further for more analysis*.

Materials & Set Up

• 1/4 of a shot glass full of your saliva
• several drops of dish soap (look for sodium laurel sulfate in the ingredients)
• a pinch of table salt (1/16 of a teaspoon)
• some contact-lens cleaning solution, meat tenderizer, or pineapple juice (optional)
• Ice-cold 120-proof+ liquor (overproof rum works well)

SAFETY NOTE:
The chemicals used in this experiment are “everyday” household items and are not particularly dangerous. Nonetheless, exercise extra caution and think twice if you decide to consume your DNA shot and ABSOLUTELY do not substitue rubbing alcohol, isopropyl alcohol, or any other non-consumable alcohol for the overproof rum we used. Besides using “denatured’ alcohol, the other potential safety concern is the dishsoap added to the mixture. A couple drops won’t hurt you, but if you are concerned about it, feel free to leave it out.

EDIT: Some DIYbioers are developing a simple gel box and a gel box on steroids . We should have some instructables put together for them before Dec 08. If you are interested in helping, please join the DIYbio google group !

step 1: Salivation.

step 2: Add a couple drops of soap

step 3: some protease…

step 4: And a pinch of salt

step 5: Pour on a layer of the rum

//

step 6: spool your DNA

//

step 7: Tastes like DNA!

MMMM – Can you taste the DNA ?

I decided to drink my DNA shot. I thought my body might resent the fact I had taken some of its DNA – the blueprint and program that defined how it grew into what it is today – and not shared any with it. Also, I wanted to see if my DNA had a particular taste.

Results : the DNA shot tastes like very potent, cheap rum. But it was one of the best drinks I can remember making.

Safety Note : be sure you are using consumable alcohol bought from a liquor store – anything else will poison you. Be safe, and think twice before you mindlessly follow directions.

More information about the chemistry of the DNA precipitation reaction and other version of the DIY DNA extraction protocol can be found here:

• Kitchen Kitchen Counter DNA lab
• The Macgyver Project: Genomic DNA extraction and Gel Electrophoresis using everyday materials
• How to extract DNA from anything living
• The Basics: How Ethanol Precipitation of DNA and RNA Works
• The Science of DNA precipitation by madsci.org

Hope you enjoyed this DIY protocol! If so, join us!
- Mac from diybio.org

http://www.instructables.com/id/5_minute_DNA_Extraction_in_a_Shot_Glass/

Posted via email from healthystealthy health hacks

J Pak Med Assoc. 2009 Apr;59(4):258-61.

Obesity induced magnesium deficiency can be treated by vitamin D supplementation.

Farhanghi MA, Mahboob S, Ostadrahimi A.

Nutritional Research Center, Tabriz University of Medical Sciences.

Posted via email from healthystealthy health hacks