Changing procedures into legends

Procedures or protocols give you a step-by-step method for carrying out your experiment. Often they are detailed lists of instructions for making up materials (solutions etc.) and what to do with these materials. When you are writing up your report, you need to change the protocol into a legend. This means you have to focus on the what you were trying to find out or measure and the conditions you used to find this out.

Roll over the highlighted text in the example procedure to see how the information is changed to a legend.

Example Procedure: A method for measuring the activity of the enzyme alcohol dehydrogenase in liver.

  • The assay has a total volume of 1.0 ml and contains 0.5 ml 0.8 M glycine, pH 9.5, 50 µl of 60 mM NAD, 50 µl of 1 M ethanol and distilled water to 980 µl.
  • Place this mix in a 1ml cuvette and zero the spectrophotometer at 340 nm. 20 µl of a liver extract is then added. This extract was prepared by homogenising 400 mg of liver with 4 ml of a homogenising buffer, containing 50 mM Tris pH 7.5 and 1 mM dithiothreitol (DTT) to stabilise the enzymes.

  • Then measure the absorbance at 340 nm every 15 s for 5 min.

  • Convert the linear rate, expressed as a change in absorbance per min, to the number of umoles of product (NADH) per min using the extinction coefficient for NADH at 340 nm, 6.2 mM-1 cm-1.

Example Legend

Liver homogenates (0.1g wet weight/ml prepared in 50 mM Tris, pH 7.5, 1 mM DTT) were assayed in 0.4 M glycine pH 9.5, containing 3 mM NAD and 5 mM ethanol. The assay contained 2 mg liver tissue/ml reaction mixture. The progress of the reaction was monitored by the increase in absorbance at 340 nm over a 5 minute time period and the reaction rate calculated using the extinction coefficient for NADH at 340 nm, ε = 6.2 mM-1 cm-1.


Follow the step by step guide on changing a procedure into a legend.

Click on each step to see the questions you must answer at each step. Then click on each question for the answer and to see how this information is used in the example legend.



Liver homogenates (0.1g wet weight/ml prepared in 50 mM Tris, pH 7.5, 1 mM DTT) were assayed in 0.4 M glycine pH 9.5, containing 3 mM NAD and 5 mM ethanol. The assay contained 2 mg liver tissue/ml reaction mixture. The progress of the reaction was monitored by the increase in absorbance at 340 nm over a 5 minute time period and the reaction rate calculated using the extinction coefficient for NADH at 340 nm, ε = 6.2 mM-1 cm-1.

Step 2:

1. What reacted with what?

Alcohol dehydrogenase in liver reacted with NAD and ethanol.

2. What was the product?

NADH was the product.

3. What is the relationship between the product and the activity of alcohol dehydrogenase?

The rate of appearance of NADH monitored by an increase in absorbance at 340 nm using the ε = 6.2 mM-1 cm-1 indicated the activity of alcohol dehydrogenase.

Liver homogenates (0.1g wet weight/ml prepared in 50 mM Tris, pH 7.5, 1 mM DTT) were assayed in 0.4 M glycine pH 9.5, containing 3 mM NAD and 5 mM ethanol. The assay contained 2 mg liver tissue/ml reaction mixture. The progress of the reaction was monitored by the increase in absorbance at 340 nm over a 5 minute time period and the reaction rate calculated using the extinction coefficient for NADH at 340 nm, ε = 6.2 mM-1 cm-1.

Step 3:

1. What is the final glycine concentration if 0.5 ml of 0.8 M glycine is used?

0.4 M glycine, pH 9.5 (the pH won't alter significantly on dilution)

2. What are the final NAD and ethanol concentrations?

3 mM NAD and 50 mM ethanol

3. What is the final concentration (g/ml) of the liver homogenate?

0.1 g liver extract per 1 ml of buffer solution - the fact that the researcher used 400 mg doesn't really matter as long as the proportions are kept constant.

4. What is the final concentration of the liver extract in the reaction mixture?

2 mg liver tissue per ml of reaction mixture.

Liver homogenates (0.1g wet weight/ml prepared in 50 mM Tris, pH 7.5, 1 mM DTT) were assayed in 0.4 M glycine pH 9.5, containing 3 mM NAD and 5 mM ethanol. The assay contained 2 mg liver tissue/ml reaction mixture. The progress of the reaction was monitored by the increase in absorbance at 340 nm over a 5 minute time period and the reaction rate calculated using the extinction coefficient for NADH at 340 nm, ε = 6.2 mM-1 cm-1.

Below are a series of extracts from two legends for measuring protein content and Beta-galactosidase activity in E.Coli. Compare the 2 extracts in each set and decide which one is most appropriate. The extracts are presented in the same sequence as they occur in the legends.

Choose only one extract in each set. You will recieve your feedback immediately.

Extract A

Six cotton-plugged conical flasks labelled A to F containing 40 ml of E. coli culture in a glycerol medium were incubated at 37 °C. The following was added to flasks A to F respectively; 2 ml of 10 mM IPTG; 0.6 ml of 27.78 mM lactose; 0.6 ml of 27.78 mM lactose and 0.5 ml of 444.44 mM glucose; 0.6 ml of 27.78 mM lactose and 1 ml of 5-FU (1000 µg/ml); 0.6 ml of 27.78 mM lactose and 1 ml of Cm (1000 µg/ml). There was no addition of compounds to flask F (control).

Extract B

E coli cultures grown to log phase in glycerol medium were treated with a number of compounds. Separate cultures were incubated for 32 min with the following compounds: 0.5 mM IPTG (defined earlier), 0.4 mM lactose, 0.4 mM lactose and 5 mM glucose, 0.4 mM lactose and 5-FU (25 ug/ml), 0.4 mM lactose and Cm (25 ug/ml) and a control with no additions.

Extract A is appropriate | Extract B is appropriate

Extract C

At various times throughout this incubation, protein content and b-galactosidase activity were measured. The protein concentration was monitored by absorbance at 600 nm using the following conversion factor: A600 * 150/1.4.

Extract D

The flasks were incubated at 37 °C. At time t= 0, 10, 20 and 35 mins following compound addition, aliquots were taken from all flasks, their absorbances measured, recorded and graphed at 600 nm.

Extract C is appropriate | Extract D is appropriate

Extract E

Beta-galactosidase activity was measured by monitoring the rate of hydrolysis of the substrate o-nitrophenol galactose (ONPG) to the coloured product o-nitrophenolate (ONP) spectrophotometrically. The assay was performed at 28oC in a buffer containing 40 mM phosphate, pH 7, 4 mM KCl, 0.4 mM MgSO4 and 20 mM b-mercaptoethanol, in the presence of ONPG at a concentration of 2.2 mM. The reaction was stopped after sufficient colour development (or 30 min) by the addition of Na2CO3 to a final concentration of 0.3 M.

Extract F

Meanwhile, at times t = 0.2.4.8.16 and 32 mins, 1 ml samples were taken from each flask and added to test tubes containing 1 ml Z-buffer, 2 drops chloroform and 1 drop 0.1% SDS to stop further β-gal induction. Each test tube was then pre-incubated at 28 °C for 5 minutes. To begin assay, 400 µM o-nitrophenol-β-D-galactopyranoside (ONPG) was added to each test tube and left to incubate at 28 °C for 10 minutes. The assays were stopped by adding 1 M Na2CO3 to the test tubes and vortexing for 10s. Absorbances of each test tube were measured at 420 nm and 550 nm and recorded.

Extract E is appropriate | Extract F is appropriate

Extract G

The final specific activity of β-gal was determined using recorded A420 and A500, protein concentrations at t = 0,2,4,8,16 and 32 minutes determined (using the A400 against time graph produced earlier) and the o-nitrophenol (o-NP) extinction coefficient, 4.5 mM-1cm-1.

Extract H

The formation of o-NP was measured at 420 nm using an extinction coefficient of 4.5 mM-1cm-1 after correcting for turbidity at 550 nm (correction factor: A420 1.3 * A550).

Extract G is appropriate | Extract H is appropriate

Read the background to the experiment for measuring the rate of muscle glycogen consumption during exercise. Then read the extracts from the procedure and choose the correct final concentrations for each component. Click the Submit button for your feedback

Is muscle glycogen metabolism affected by fitness?

Glycogen, a polymer of glucose, is stored in muscles as a ready source of fuel during exercise. This study investigates the relationship between fitness and the rate of muscle glycogen consumption during exercise.

Study Design: Eight subjects (19-22 years), 4 male and 4 female, with varying levels of fitness were chosen. Fitness levels were assessed by measuring the O2 consumption during a 5 min period of maximum sustainable exercise. Subjects then cycled for 2 h; muscle biopsies were taken at 30 min intervals during the exercise period.

Extract 1

Buffer

Weigh out 10.4 g sodium phosphate (Na H2PO4. H2O), Mw = 138). Dissolve the NaH2PO4 in 450 ml water. Bring pH of solution to 7.4 with 10 M NaOH (needed about 2 ml). Make volume up to 500 ml with water. Store in fridge.

0.076 M | 0.15 M | 10.4 M | 150 mM
Iodine 0.5/500mL = 1g/L
				 = 0.1g/dL
                 = 0.1% (w/v)
or

1g/L = 1
254

false

Extract 2

KOH for tissue digestion

Weigh out 2.8 g KOH (Mw = 56) and add to 40 ml of water. Do this on ice as it is a very exothermic reaction. Make volume up to 50 ml with water. Store at room temperature.

2.5 mM | 0.05 M | 0.56 g/ml | 50 mM

true

false

Extract 3

Ethanol for glycogen precipitation: Take 70 ml ethanol and add 30 ml water. Mix. Store at room temperature.

70% v/v | 70 mM | 2.7 mM | 40%v/v

true

false

Extract 4

Iodine Reagent

Dissolve 0.5 g iodine (Mw = 254) in about 380 ml water. Add 50 ml of 1 M potassium iodide and 25 ml 1 M calcium chloride. Make up to a total volume of 500 ml with water. Store at room temperature.

0.1% (w/v) iodine,100 mM KI and 50 mM CaCl2
0.2% (w/v) iodine, 200 mM KI and 100 mM CaCl2
0.1% (w/v) iodine, 100 mM KI and 50 mM CaCl2
4 mM iodine, 100 mMKI, 50 mM CaCl2
false
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