My middle school class tried DNA extractions from dried split peas and cheek cells for a lab this week, and the experiments went rather well.

dna-lab-2689s — Strands of DNA rising from the interface between crushed split pea “sludge” and rubbing alcohol. Bubbles trapped beneath the strands make for interesting convective-like patterns.

Split Pea DNA

For the split peas, we followed the Learn.Genetics lab. I blended the peas in some salty water at the front of the class (“Don’t you need a lid for that blender?”), and filtered it. This gave about 120 ml of filtrate. Then I added in 3 tablespoons of dish soap to the filtrate. The soap to breaks down the cell membranes and nuclear membranes because they are made of lipids (fats).

dna-split-pea-residue-2697 — Split pea residue left behind after filtering.

I shared out the resulting green liquid to the four groups. Each student was able to get a fair amount into a test tube so they could complete the lab as individuals.

Disintegrating the cell and nuclear membranes with soap exposed the DNA, but the long DNA molecules tend to be coiled up around proteins. Each student added a pinch (highly quantified I know) of meat tenderizer to their test tube to break down the protein and allow the DNA to uncoil. Enzymes are biological catalysts, large complex molecules that accelerate chemical reactions, the breakdown of proteins in this case, without breaking down themselves, so only a little is needed.

Finally, each student carefully poured rubbing alcohol into the test tube. I had to demonstrate how to tilt the test tube while alcohol was being poured into it so that the alcohol would not mix in with the pea soup but, instead, form a layer at the top, since the alcohol is less dense than the suspension of split peas in salty water.

dna-lab-2692s — When the DNA is mixed in with the split pea filtrate (right) it becomes a little harder to distinguish. On the left you can see that the clear rubbing alcohol floats on top of the denser split pea/water/salt mixture.

If it was done carefully enough, the DNA would precipitate at the boundary between the two liquids. If not, the DNA would still precipitate, but it would be mixed in together with the green soup and be harder to distinguish.

Either way, however, students could see the long strands of DNA, and fish them out with glass rods.

dna-lab-2688s — Strands of DNA on a glass rod.

Human DNA

The human DNA extraction procedure is well demonstrated by the NOVA video. One student who missed the split pea lab did this experiment instead because it’s faster. It does not require blending to crush the cells, nor does it need the meat tenderizer enzyme.

Although this procedure produces a lot less DNA — after all, you’re only getting a few loose cells from the insides of your cheeks — the strands are still visible. And it’s YOUR DNA.

dna-human-2715 — A few strands of human DNA (belonging to an individual who asked to be referred to as “Suzanne”) in a test tube. The rubbing alcohol is dyed blue for visual contrast.

Since I instructed the class on how to use the microscopes last month, one student wanted to see what his DNA looked like under the microscope. An individual DNA molecule is too small to see, but the strands we have are bunches molecules that are visible. They just don’t look like very much.

dna-2719 — DNA strands under the microscope.

Abstract

I punched a small hole (about 1mm radius) in a one gallon plastic bottle and had my students measure the rate at which water drained. Even though the apparatus and measurement technique was fairly rough, we were able to, with a little calculus, determine the equation for the height of the water in the bottle as a function of time.

calculus_jug_1110 — Figure 1. A student uses a stopwatch to measure the outflow rate of water from the plastic bottle.

Introduction

Questions about water draining from a tank are a pretty common in calculus textbooks, but there is a significant difference between seeing the problem written down, and having to figure it out from a physical example. The latter is much more challenging because it does not presuppose any relationships for the change in the height of water with time; students must determine the relationship from the data they collect.

The experimental approach mimics the challenges faced by scientists such as Henry Darcy who first determined the formulas for groundwater flow (Darcy, 1733) almost 300 years ago, not long after the development of modern calculus by Newton and Leibniz (O’Conner and Robertson, 1996).

Procedure

I punched a small hole, about 1mm in radius, in the base of a plastic, one-gallon bottle. I chose this particular type of bottle because the bulk of it was cylindrical in shape.

Students were instructed to figure out how the rate at which water flowed out (outflow rate) changed with time, and how the height of the water (h) in the bottle changed with time. These relationships would allow me to predict the outflow rate at any time, as well as how much water was left in the container at any time.

Data collection:

To measure height versus time, we marked the side of the bottle (within the cylindrical region) in one centimeter increments and recorded the time it took for the water level to drop from one mark to the next. There were a total of 11 marks.
To measure the outflow rate, we intercepted the outflow using a 25 ml beaker (not shown in Figure 2), and measured the time it took to fill to the 25 ml mark.

Results

Time elapsed since last measurement	Height of water in container	Time to fill 25ml beaker
Δt (s)	h (cm)	t_f (s)
0.0	11	6.4
45.5	10	7.1
52.3	9	8.5
43.1	8	7.1
56.1	7	7.8
60.6	6	8.3
57.6	5	8.9
65.0	4	9.9
72.8	3	11.2
76.7	2	13.0
91.1	1	14.8
122.0	0	17.3

Table 1: Outflow rate, water height change with time.

To analyze the data, we calculated the total time (the cumulative sum of the elapsed time since the previous measurement) and the outflow rate. The outflow rate is the change in volume with time:

$\text{outflow rate} = \frac{volume}{time} = \frac{dV}{dt} = \frac{25 ml}{t_f}$

So our data table becomes:

Time	Height of water in container	Outflow rate
t (s)	h (cm)	dV/dt (ml/s)
0.0	11	3.91
45.5	10	3.52
97.8	9	2.94
140.9	8	3.52
197	7	3.21
257	6	3.01
315.1	5	2.81
380.1	4	2.53
452.9	3	2.23
529.6	2	1.92
620.7	1	1.69
742.7	0	1.45

Table 2. Height of water and outflow rate of the bottle.

The graph of the height of the water with time shows a curve, although it is difficult to determine precisely what type of curve. My students started by trying to fit a quadratic equation to it, which should work as we’ll see in a minute.

h-v-t — Figure 3. The decrease in height with time is not a straight line (is non-linear).

The plot of the outflow rate versus time, however, shows a pretty good linear trend. (Note that we do not use the first three datapoints (in Table 2), which we believe are erroneous because we were still sorting out the measuring method.)

Although I’ll note here that the data should ideally be modeled using a square root equation (Torricelli’s Law), that is beyond the present scope of the problem (we’ll try that tomorrow as a follow exercise).

Plotting the data in Excel we could add a linear trendline.

q-v-t — Figure 4. The outflow rate decreases linearly with time.

For the linear trendline, Excel gives the equation:

$y = -0.0035 x + 3.9113$

The y-axis is outflow rate (the change in volume with time), and the x axis is time (t), so the linear equation becomes:

$\frac{dV}{dt} = -0.0035 t + 3.9113$

Notice that this is a differential equation.

To determine the rate of at which the height of water in the container is changing, we need to recognize that the container is cylindrical in shape, and the volume (V) of a cylinder depends on its radius (r) and height (h):

$V = \pi r^2 h$

which can be substituted into differential in the rate equation:

$\frac{d(\pi r^2 h)}{dt} = -0.0035 t + 3.9113$

since π and the radius (r) are constants (since the jug’s shape is a cylinder), they can be pulled out of the differential:

$\pi r^2 \frac{dh}{dt} = -0.0035 t + 3.9113$

Dividing through by πr² solves for the rate of change of height:

$\frac{dh}{dt} = \frac{-0.0035 t + 3.9113}{\pi r^2}$

Isolating the coefficients gives:

$\frac{dh}{dt} = \frac{-0.0035}{\pi r^2} t + \frac{3.9113}{\pi r^2}$

This equation should give the rate at which the height changes with time, however, if you look at it carefully you’ll realize that for the time range we’re using (less than 800 seconds) the value of dh/dt will always be positive. We correct this by recognizing that the outflow rate is a loss of water, so a positive outflow should result in a negative change in height, therefore we rewrite the equation as:

$-\frac{dh}{dt} = \frac{-0.0035}{\pi r^2} t + \frac{3.9113}{\pi r^2}$

which gives:

$\frac{dh}{dt} = \frac{0.0035}{\pi r^2} t - \frac{3.9113}{\pi r^2}$

Now comes the calculus

Now, we can use this rate equation to find the equation for height versus time by integrating with respect to time:

$\int \frac{dh}{dt} dt = \int \left( \frac{0.0035}{\pi r^2} t - \frac{3.9113}{\pi r^2} \right) dt$

to get:

$h = \frac{0.0035}{2 \pi r^2} t^2 - \frac{3.9113}{\pi r^2} t + c$

And all we have to do to the find the constant of integration is substitute in a known point, an initial value. As is often the case, the best point to use is the starting point where t=0 makes the rest of the calculations easier. In our case, when t=0, h=11:

$11 = \frac{0.0035}{2 \pi r^2} (0)^2 - \frac{3.9113}{\pi r^2} (0) + c$

so:

$c = 11$

And our final equation becomes:

$h = \frac{0.0035}{2 \pi r^2} t^2 - \frac{3.9113}{\pi r^2} t + 11$

which is a quadratic equation as my students guessed before we did the calculus.

Does it work?

You will notice that in the math above, we never use the height data in determining equation for height versus time; all the calculations are based on the trendline for the outflow rate versus time.

As a result, we can compare the results of our equation to the actual measurements to see if our calculations are even close. Remarkably, they are.

h-v-t-model-v-measured — Figure 5. The results from our equation (modeled) match the measured heights so well, the data points are difficult to distinguish on the graph.

Discussion

Despite all the potential for error, particularly, our relatively crude measurement techniques, and the imperfect cylindrical shape of our plastic bottle, the experiment went remarkably well.

Students found it quite challenging, and required some assistance even though this is a problem they have seen before in their textbook.

The problems in the textbook use Torricelli’s Law, which should much better describe the draining of a tank than the linear equation we find for dV/dt.

Torricelli’s Law:

$\frac{dV}{dt} = a \sqrt{2gh}$

where a is the area of the outlet hole, and g is the acceleration due to gravity.

In our actual experiment it is difficult to tell that a square root function would work better. Excel does not have an option for matching a square root function, so the calculations would become more involved (although it could be set up using Excel’s iterative solver or Goal Seek function).

Conclusion

Our experiment to use calculus to determine the rate of change of the height of water in a leaking plastic water bottle was a successful exercise even though the roughness of the data collection did not permit identification of the square root law for leakage.

Tag: experiment

DNA Extraction Labs