Teacher's Guide... Micromeasurement

The reactions required for genetic analysis at the molecular level require VERY small amounts of DNA, reagents, and enzymes. The two most common units of liquid measurement that are used in these laboratory experiments are the milliliter (mL) and the microliter (uL).

1 mL = 1/1000 liter	1000 mL = 1 liter
1 uL = 1/1,000,000 liter	1,000,000 uL = 1 liter

Use of Micropipets:

Digital micropipet

There are three important DON'TS when using a digital micropipette:

1. DON'T attempt to use the pipet without a tip in place. This can ruin the precision piston that determines the volume of the fluid.

2. DON'T lay down a pipet that has a filled tip. Fluid in the tip could run back into the pipet and ruin the precision piston.

3. DON'T let the delivery button (control button) snap back after withdrawing or delivering fluid.

To use a digital pipet:

1. Rotate the delivery button to the desired volume.

2. Push the pipet firmly into the proper size micropipet tip.

3. While withdrawing or expelling liquid, hold the vessel and pipet at nearly eye level. It is important to watch while you pipet.

4. Hold the pipet close to the vertical while pipeting.

To withdraw a sample:

1. Depress the button to the first stop and hold the button in that position. Dip the tip of the pipet into the fluid to be pipeted and slowly release the button to draw the fluid into the tip.

2. Slide the tip of the pipet along the side wall of the reagent tube to knock off excess fluid that might have remained on the outside of the tip.

To expel a sample into a reagent tube:

1. **Touch the pipet tip to the inside wall of the reaction tube (microcentrifuge tube) into which the sample will go. This will help create a capillary effect which should help draw the fluid out of the pipet tip.

2. To expel the sample, slowly depress the button to the first stop. Wait a second or two and then press on to the second stop to blow out the last bit of fluid. Continue to hold the button in the second position as the pipet tip is withdrawn from the tube.

3. To eject the tip (if the pipet has this feature), press the ejector button down while the pipet is held over a beaker designated as the receptacle for used tips.

4. To prevent contamination of the reagents used in the laboratory, follow these guidelines:

• Always add appropriate amounts of a single reagent sequentially to all reaction (microcentrifuge) tubes.
• Release each reagent drop onto a NEW LOCATION on the inside wall of the reaction tube. In this way you can continue to use the same tip to pipet a number of samples of a single reagent into different reaction tubes.
• Use a fresh pipet tip for each new reagent you pipet.

Microcapillary pipet

Capillary micropipettes are capillary tubes that are marked to accept a specific microquantity of fluid. Usually they employ a thin metal rod for suction. If due care is used, they are an accurate and economical way to apply samples for use in electrophoresis. (Piston Type)

1. Insert the metal piston (wire) nearly all the way into the capillary tube. (Try not to expose the piston to the sample.)

2. Dip the tip of the capillary tube into the fluid, pulling back on the wire piston to fill the tube. Draw up to the line.

3. To expel, push down on the wire piston. Touch the tip of the capillary tube to the inside wall of the microcentrifuge (reaction) tube. (See "1. **", above.)

To prevent contamination of the reagents used in the laboratory, follow these guidelines:

• Always add appropriate amounts of a single reagent sequentially to all reaction (microcentrifuge) tubes.
• Use a fresh pipet tip for each new reagent you pipet.

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Student Analysis Sheet

In protein separation by SDS polyacrylamide gel electrophoresis, migration is determined not by intrinsic electrical charge of polypeptides but by molecular weight. Sodium dodecylsulfate (SDS) is an anionic detergent that denatures proteins and confers a negative charge to the polypeptide in proportion to its length. Treatment of proteins with SDS and a reducing agent causes the individual polypeptides to become negatively charged, with equal charge per unit length. We then can determine the molecular weights of polypeptides by running a gel with standard proteins of known molecular weight along with the unknown polypeptides. A linear relationship exists if the logarithm of the molecular weights of standard proteins are plotted against their respective relative mobilities (Rf). To determine the relative mobility (Rf) of a protein, divide its migration distance from the top of the gel to the center of the protein band by the migration distance of the tracking dye from the top of the gel.

	distance of protein migration
Rf =
	distance of tracking dye migration

A standard curve is constructed by plotting the Rf values of the standard polypeptides against the logarithm of their molecular weight. The Rf values of each unknown polypeptide is determined in the same way. The log of molecular weight is read directly from the standard curve; the antilog is the molecular weight.

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Experimental Results

Figure T1

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Experiment Analysis:

1. Compute the log of each marker fragment presented in the Table 1.

2. Measure the distance (in mm) from the sample well to each fragment. Try to be consistent: measure from the well to the middle of the fragment band. Record each in Table 1, in order, beginning with the band closest to the sample well.

3. Measure the distance from a sample well to the end of the gel.

Table T1

Data Table
Low Molecular Weight Protein Markers
	Protein	Molecular Weight ( Daltons)	Log	Distance Migrated (mm)	R f
Fragment 1	Bovine Serum Albumin	66,000	4.82	21	0.28
Fragment 2	Albumin, Egg	45,000	4.65	27	0.36
Fragment 3	Carbonic Anhydrase	29,000	4.46	38	0.51
Fragment 4	Trypsin Inhibitor	20,000	4.30	51	0.68
Fragment 5	Lysozyme	14,000	4.15	60	0.8

4. Calculate the Rf of each fragment; record data in Table 1. NOTE: The distances from individual wells to the end of the gel may not the same. Therefore, the Rfs should be calculated based on well distances measured from individual lanes.

5. On a sheet of graph paper, construct a standard curve plot using the molecular weight protein marker electrophoretic data. Plot Rf (horizontal axis) against the logarithm of their molecular weight (vertical axis).

6. From your standard curve you can determine the molecular weight of the unknown polypeptides.

   Note: The gel is 75 mm long. Therefore, Rf values are calculated as migration distances (in mms) divided by 75 mm.

Teacher's Notes: The experimental data presented in the table is taken from the gel photograph (Figure T1). Individual student results may vary from these published results. Student data should approximate this data; the overall relationships of band placement should be consistent with the data presented in the table.

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