The exercise of power is determined by thousands of interactions between the world of the powerful and that of the powerless, all the more so because these worlds are never divided by a sharp line: everyone has a small part of himself in both - Vaclav Havel
Briefly put, the protocol constitutes four separate steps:
Making lightly salted "pea cell soup".
Breaking down the cells and nucleus membranes, thereby releasing the raw DNA into the mix, by adding washing-up liquid.
Breaking down the proteins onto which the DNA is coiled, by adding an enzyme to the mix.
Separating the freed DNA by means of alcohol.
I had to make a few small changes to the recipe though:
Having no split peas at hand I used frozen ones and substituted the recommended 100 ml of the former by 100 g of the latter (a bit of a mistake).
Having no "meat tenderizer" at hand (it seems they don't sell that stuff in Europe, at least not in retail), I followed the suggestion of trying pineapple juice, which contains the enzyme Bromelain, also found in meat tenderizers. I made freshly squeezed pineapple juice by straining some 10 ml of finely chopped pineapple through a tea-strainer.
Having no rubbing alcohol, I replaced it with (purple) methylated spirits, which is essentially methanol.
Not having the slightest idea how much Bromelain my juice contained, I decided to run a few tests at once. And so after completing steps 1 and 2, I added respectively 1 ml, 2 ml and 3 ml of pineapple juice to three test-tubes (from my daughter's chemset, every home should have one!), each containing the same amount of treated pea soup (about 3 ml).
Very little patience was rewarded almost immediately, as a white, flocular material started to appear in the zone separating the pea soup and the alcohol! It's slightly eerie to see the stuff that is so vital to life appear before your very own eyes...
A few conclusions could also be drawn:
The total amount of DNA (plus some RNA, as this is so-called 'genomic DNA') seemed a lot smaller "than in the picture". This I believe is partly due to my ill-advised substitution of the recommended 100 ml of split peas, by 100 g of frozen peas: split peas are a lot more dense than frozen or fresh peas, so a 100 ml of splits probably weighs up to 150 to 200 g. My concoction simply didn't contain as much DNA as planned...
As it rose through the methanol, the DNA showed its stringy nature but it wasn't quire as stringy as I expected: too much stirring (these macromolecules are fragile) or too high expectations?
The amount of DNA was clearly proportional to the amount of pineapple juice used. At first glance it would be tempting to attribute this to the greater quantity of enzyme present but that doesn't necessarily make sense. The pineapple juice was a thick and non transparent liquid, probably closer to "pineapple cell soup" than an actual Bromelain solution and thus more DNA will have been present in the test-tubes containing the higher amounts of pineapple juice. An additional test using 3 ml of pineapple juice with a reduced amount (about 2 ml) of pea cell soup yielded about the same amount of DNA as the 3/3 combination, indicating I was also extracting pineapple DNA and not just pea DNA. One way to find out would be to filter the pineapple juice...
In a next session I'll be testing some of these conclusions/hypotheses by means of a small factorial experimental design using split v. frozen, filtered v. unfiltered pineapple juice and 1 ml v. 3 ml of pineapple juice as factors. Using a saturated randomised experiment matrix, I can evaluate the effects of these three factors by means of just four runs (and as luck would have it, I have exactly four test-tubes!)
The procedure can be applied to just about any living matter and I'll be testing it on one of my closest enemies soon enough (just kiddin', just kiddin'!) but peas are a good source because they are seeds and contain lots of the stringy stuff. And most of Gregor Mendel's groundbreaking work on the laws of genetic inheritance was also done on peas and pea plants. So, let's hear it for the father of modern genetics and his trusted friend, the good old pea! But if you can't be bothered with DNA or genetics, here's some recipes for tasty pea soup... Update: the results of the additional tests can be found here.
I decided to test some of these conclusions/hypotheses by means of a small factorial experimental design using split v. frozen, filtered v. unfiltered pineapple juice and 1 ml v. 3 ml of pineapple juice as factors. Using a saturated randomised experiment matrix, I can evaluate the effects of these three factors by means of just four runs.
Not being able to find split peas, I settled for dried marrowfat peas, a British favourite used in preparing mushy peas (a common accompaniment to fish 'n chips). I used 100 g of these but due to their extreme water absorption (I soaked them overnight, as I did the frozen peas), I had to add another 50 ml of water to the standard 200 ml, in order to be able to blitz the peas into "pea cell soup". So the comparison below is really between 100 g of frozen peas/200 ml of water on the one hand, and 100 g of dried marrowfat peas/250 ml of water on the other hand. All other factors, such as the amount of salt, the amount of washing-up liquid and working time, were kept as constantly as possible. The "pea cell soup" obtained from the marrowfat peas was much thicker and a lighter shade of green with a bit of froth (foam) on top (see also below).
The pineapple juice was obtained by blitzing half of a baby pineapple, passing the fruit paste through a tea trainer and filtering half of that using a funnel and some kitchen towel (tissue). The filtered pineapple juice was slightly opaque and thin, the strained pineapple juice was thick and completely non-transparent.
The experiments were run almost simultaneously, using about four ml of the treated pea soup (recipe)
Here's the experiment matrix and the response variables obtained:
Run>>>>> Peas>>>>>> Juice>>>>> Juice>>>> DNA (rating 1- 5)
#1>>>>>> Frozen>>>>> Unfiltered >3 ml>>>> 1
#2>>>>>> Frozen>>>>> Filtered>>> 1 ml>>>> 1
#3>>>>>> Dried>>>>>> Unfiltered>> 1 ml>>>> 4
#4>>>>>> Dried>>>>>> Filtered>>>> 3 ml>>>> 5
Thanks to a "free gift" from DELL, I'm the owner of what must be the crappiest digital camera in the universe, so bear with me on the image below. The tubes are arranged for left to right as #1, #2, #3 and #4. The photo below was taken approximately 5 min after adding the methylated spirits. Although the images make it difficult to discern the amount of DNA separated, I used a visual (direct, non-photographical) ranking system as my response variable, on a scale from 1 to 5, with ranking 5 indicating the highest amount of DNA and 1 the lowest. This system is of course subjective and imprecise but at the time it was the best I could do: the amounts would not lend themselves to weighing with my electronic scales, which only have a 0.1 g precision.
In tube #1, an opaque disc of DNA could be seen, risen to the top of the alcohol phase. In tube#2, a similarly faint disc was about to rise to the top too. I ranked both as 1.
For tubes #3 and #4, please note that the foam mentioned before had settled on top of the alcohol phase and this foam is not indicative of the amount of DNA. But between the pea soup/alcohol separation and the foam, it can be clearly seen that much more DNA has gathered there than in the case of runs #1 and #2. I ranked #3 as a 4 and #4 as a 5.
To calculate the effects of the factors, simply add the response variables (DNA) of both runs where the factor was at one level (1), add the response variables (DNA) of both runs where the factor was at the other level (2), calculate (2) - (1) and divide by 2.
For example in the case of the type of peas, (1) = 1 + 1 = 2, (2) = 4 + 5 = 9, (2) - (1) = 7 and the effect of switching from frozen to dried peas thus equals 7/2 = +3.5.
Similarly the effect of switching from unfiltered to filtered pineapple juice was +0.5 and the effect of switching from 1 ml to 3 ml of pineapple juice was -0.5. The average of all four runs was 2.75.
So, let's draw some conclusions:
Peas: the effect of the type of peas, predictably perhaps, was very clear; dried peas yield more of the stringy stuff, presumably because they contain more cell matter and less water.
Filtering: due to the low resolution of the design, an effect value of +0.5 is probably not statistically significant. It's also in breach of a previous assumption, namely that using pineapple cell pulp, instead of filtered pineapple juice, would introduce more DNA to the mix and increase yield. It's entirely possible though that using unfiltered pineapple juice in conjunction with a DNA-poor cell soup (i.e. using frozen peas) could slightly increase the yield of DNA (as was previously observed).
Amount of pineapple juice: again, due to the low resolution of the design, an effect value of -0.5 is probably not statistically significant. In practice this would mean that the required amount of pineapple juice is probably less than 1 ml and that a few drops would do the trick. Enzymes are bio-catalysts and very effective at what they do: a small amount probably suffices.
And so, I'll probably be running one more test, this time using split peas and determining the minimum needed quantity of filtered pineapple juice...
I while ago I stumbled on a science site that expanded on a simple procedure to extract DNA from living matter (or from anything that once was alive and hasn't decayed too much). Yesterday I decided to put it to the test. You can find a complete layman's version here, including some enlightening schematics. It's applied to peas.
