Grow grow grow!

So I checked up on my plants recently and was very pleased to find that they have been growing quite nicely since my last update:

15s population

30s 

60s and 90s

The time is ripe for assays!

I'm nearly out of reagents from my first kit, so I ordered the second kit last week, and hopefully, the package will be coming soon. (But evidently, not soon enough!)

Control Conundrums

Okay so as it turns out, the past two weeks I've been busy standardizing my procedure still, making sure that a) my experimental procedure is repeatable b) my procedure actually works.

I repeated the cornstarch control experiment a second time:

As you can see, this trendline, compared to that of the previous control graph, differs markedly... THE SLOPE IS NEGATIVE, which doesn't make any sense! #whyisthismylife

When I first finished my calculations, I had a mini panic-attack - I checked and rechecked my number chugging, and found nothing wrong. In the midst of my alarm, a sudden brainwave - I had my lab notebook!

So, I keep this marble lab notebook in the prep room over the course of my experiment where I attempt to record in as much detail as possible my procedures - what I actually carried out as opposed to what I typed up to carry out. If I had many any changes to the procedure that might have accounted for this drastic difference in data trends, this was the place where I would have written it down.

I ran and grabbed my notebook, flipped through the pages (some of them stained with mystery liquids), and Voila! It all started coming back to me.

In this particular procedure, I had massed the sample weights with a different method - rather than using the weigh paper method, I first tared the test tubes themselves, then measured out the correct amount of cornstarch into the tubes and massed that. Because it was difficult to add and subtract precise amounts of cornstarch, I had to redo one massing. I had to wash out the tube, and so there were minute droplets of water left over that never completely dried out - this would have made a difference, possibly substantial, in the actual and measured mass of the cornstarch samples.

As if that one change were not enough, because of time constraints in this round (last time, I had done a good two-thirds of the procedure afterschool, whereas this time, I had to cram as much as I could within my 1 hour period in school), I paused the experiment just before proceeding with the starch digestion (right after boiling the alpha-amylase reaction for 5 minutes). Perhaps keeping the reaction in the refrigerator overnight before proceeding directly to the next step might have had some bearing on the differences in the data. 

So Hyped!

So I'm back from my winter break, and next week, I will be beginning the first round of my actual experiments in assaying my duckweed samples. I will have to grind up representative duckweed fronds to carry the procedure out as I've been doing for the past few weeks...super excited!

Calculation Results

Alright, so down to some number-crunching!

The magic formula I used for calculating percent starch was:

% Starch = [(∆Atest )(900) ]/  [(∆Astd)(sample weight in mg)]

(I won't bore you with details on where the 900 came from but it involves the multiplication of molecular weight of starch monomers, initial sample volumes from glucose sample volume assays, dilution factors, and whatnot.)

Okay. The values for the 1 mg sample, 5 mg sample, and 10 mg sample turned out to be 24.3%, 33.7%, and 56.7%.

Plotted on a graph, the relationship turns out to be somewhat linear, which is a good and expected:

Next week I will repeat the standardization experiment to make sure everything is correct (and to make sure it is repeatable), before moving on to the real samples.

Pink is My Favorite Color (Day 2 Standardization Continued)

Check out these beauts!

Fig. 1 (From left to right): Standard Blank, Standard, Reagent Blank, Cornstarch Samples "1", "5", "10"

The intensity of the pink color pictured above is proportional to the original glucose percentage. I got absorption readings of 0.02, 1.2, .185, .22, .41, and .95, respectively (at 540nm).

Also, this was my work area:

Fig. 2

...Cozy, no?

(That labeled bottle on the left is the sulfuric acid solution! And the paper on the right - that's my procedure bible.)

I'm going to take these measurements home and plug them into my calculations, and see what I'll get for the percent starch. 

This is so cool!!! :)

Day 2 of Starch Assay Standardization

So today I was back in 201P finishing up the remainder of last night's procedure. The first thing I did was turn on the Spectrophotometer (Spec 20) and let it warm up for 15 minutes. Then I retrieved my samples and pipetted the solutions - my Standard Blank (distilled water), Glucose Standard (water + the glucose that came in the kit), Reagent Blank (the blank from the Starch digestion - contains only the reagents), and the 3 cornstarch samples - into their respective cuvettes.

This step was all about timing. If we let the reaction for each sample go on for differing amounts of time, we aren't going to be evaluating them on the same standard. So upon adding the Glucose Assay Reagent to each sample, I had to incubate each tube for exactly 30 minutes in a 37˚C water bath; then, I had to stop each reaction by adding 2ml of the 12N Sulfuric Acid solution.

I figured the easiest, most efficient way I could do this was by adding the Glucose Assay Reagent to the first tube (standard blank) mix, and then put it in the incubator; wait 1 minute, before adding the reagent to each subsequent tube. Then, when time was up for the first tube, I would add the H2SO4, take that out, and then wait another minute; that's how I knew the 30 minutes was up for the second tube, and so on and so forth.

But I'm not going to lie - it was pretty nervewracking. I don't think my eyes ever left the screen of my i-phone timer.

An Elaboration

I thought I'd take a moment to clarify what this procedure is all about.

Essentially this starch assay is a reaction in 3 steps: 

I stopped right after the first reaction; after the fourth reaction, I will be left with samples that will be pink in color, in varying shades. I will be using a spectrophotometer to measure the absorbency of the samples, and this measurement will be proportional to how much starch is in each.