Lab 1

Question 1

What is a micropipettor

Answer 1

a micropipettor is a precision pump fitted with a disposable tip. The volume it can accurately measure is minuscule (as little as 1 microlitre uL)

Question 2

how does a micropipettor work

Answer 2

The volume of air space in the barrel is adjusted by screwing the plunger further in or out of the chamber. Depressing the plunger displaces the specified volume of air from the chamber and releasing the plunger creates a vacuum, which draws an equal amount of fluid into the tip. Depressing the plunger again then expels the withdrawn fluid.

Question 3

what is the range of a P10 Micropipette

Answer 3

A P10 Micropipette has a volume range of 0.5-10 uL and uses a white tip

Question 4

what is the range of a P20 Micropipette

Answer 4

A P20 Micropipette has a volume range of 2-20 uL and uses a yellow tip

Question 5

what is the range of a P200 micropipette

Answer 5

A P200 Micropipette has a volume range of 20-200 uL and uses a yellow tip

Question 6

what is the range of a P1000 micropipette

Answer 6

A P1000 Micropipette has a volume range of 200-1000 uL and uses blue tips.

Question 7

What are 8 precautions when using micropipettors

Answer 7

• Always hold vertically
• Never rotate the volume adjustor beyond the upper and lower range of the pipette
• Never use without a disposable tip in place
• Never reuse a tip that has been used to measure a different reagent
• Never immerse the barrel of the micropipettor in fluid
• Never let the pinger snap back after withdrawing or expelling fluid
• Never invert or lay the micropipettor down with a filled tip
• Never flame the tip of the micropipettor

Question 8

how do you set the volume on a micropipette

Answer 8

To set the volume on a micropipette:

The volumeter consists of 3 or 4 dials, which are used to set the volume of the liquid to be transferred. They are read from top, which is most significant, to the bottom digit, which is least significant.

The volume is adjusted by rotating the black adjustment ring on the Gilson pipettes or unlocking the lock level and rotating the thumb knob and then relocking it once the volume is set. Rotating clockwise reduces the volume.

Question 9

what should you do with the reagent tube when pipetting

Answer 9

When withdrawing or expelling fluid, always hold the reagent tube firmly between thumb and forefinger. Hold the tube nearly at eye level to observe the change in fluid level in the pipette tip.

Question 10

what should you do with the tube when pipetting

Answer 10

Do not pipette with the tube in the test tube rack. D

Do not have another person hold the tube while you are pipetting.

Each tube must be held by the tube body during manipulation.

Question 11

how should you grasp the micropipettor for best control

Answer 11

For best control grasp the micropipettor in your palm and wrap your fingers around the barrel; work the plunger with your thumb. Hold almost vertical.

Question 12

what are the two stops of a micropipettor

Answer 12

Most micropipettors have a two-position plunger with friction “stops”. Depressing to the first stop measures the desired volume. Depressing to the second stop introduces an additional volume of air to blow out any solution remaining in the tip

Question 13

how do you withdraw the sample form a reagent tube

Answer 13

To withdraw the sample from a reagent tube:

• depress the plunger to the first stop and hold in position. Dip the tip into the solution to be pipetted and draw the fluid into the tip by gradually releasing the plunger. Be sure the tip remains in solution.
• Check there is no air space at the end of the tip or air bubbles within the sample.

Question 14

How do you expel the sample into a reaction tube with a micropipette

Answer 14

To expel the sample into a reaction tube:

• Touch the tip of the pipette to the inside wall of the reaction tube into which the sample will be emptied. This creates a capillary effect
• Slowly depress the plunger to the first, then second stop. Hold in depressed position
• With the measurement plunger depressed, slid the pipette out of the reagent tube, to avoid sucking any liquid back into the tip
• Return the plunger to its released position. Do not let the plunger snap back.
• Eject tip into waste jar by ejection button
• Poll the sample by sharply tapping the tube on the bench top or pulsing it in a centrifuge

Question 15

how do you prevent cross contaminations. of reagents

Answer 15

To prevent cross contaminations of reagents.

• Always add appropriate amounts of a single reagent sequentially to all reaction tubes
• Use a fresh tip for each new reagent to be pipetted

Question 16

how do you prepare a standard curve

Answer 16

To prepare a standard curve, one measures the absorbance of a series of coloured solutions of known concentration. The plot of absorbance against concentration produces a straight-line relationship, which is called a standard curve. One can then use the linear relationship to interpolate the concentration of an unknown solution of the same compound from its measured absorbance.

Question 17

what are standard curves frequently used for

Answer 17

Standard curves are frequently used to measure the concentration of an intracellular component which are often colourless. For this reason, prior to spectrophotometry it must be reacted with a specific dye called a chromogen that will convert it to a coloured product.

Question 18

how can you express very large numbers in this lab

Answer 18

In this lab exponential (logarithmic) expression can be written in two forms:

• 5.3 x 10^5
• 5.3 E5

Be consistent!

Question 19

what are sig figs

Answer 19

The number of significant figures in a measurement is equal to the number of digits that are known with some degree of confidence, plus the last digit. Ex: 2.531 has four significant digits, where 2.53 is known with some degree of confidence plus the 1

Question 20

how many sig figs should you use

Answer 20

You should present your data with a reasonable level of precision.

As we improve the sensitivity of the equipment used to make a measurement, the number of sig figs increases. The degree of precision depends on the instrument used.

Use the number of significant figures equivalent to the level of precision obtained by the least precise of your measuring devices. If you are not sure whether a digit is significant, assume that it is not.

Question 21

what are length measurements

Answer 21

Length:

1 meter (m) = 1000 millimeters (mm)

1 millimeter (mm) = 1000 micrometers (um)

1 micrometer (um) = 1000 nanometers (nm)

Question 22

what are the mass measurements

Answer 22

Mass

1 gram (g) = 1000 milligrams (m)

1 milligrams (mg) = 1000 micrograms (ug)

1 micrograms (ug) = 1000 nanograms (ng)

Question 23

what are the volume measurements

Answer 23

Volume

1 Liter (L) = 1000 milliliter (mL)

1 milliliter (mL) = 1000 microliter (uL)

1 microliter (uL) = 1000 nanoliter (nL)

Question 24

what is a solution

Answer 24

A solution is a random mixture of a soluble or ionizable molecules, the solute, in a liquid solvent. The solvent in living cells is always water, so the solutions in cells are said to be aqueous.

Question 25

what is solubility

Answer 25

Each solute molecule has its unique degree of solubility in water, and this is a temperature-dependent phenomenon - solubility increases with temperature.

Question 26

what is a saturated solution

Answer 26

A solution that contains the maximum amount of dissolved solute at a given temperature is said to be saturated. Biological solutions are usually very dilute, and almost never reach saturation.

Question 27

what does KE do for solute molecules

Answer 27

The kinetic energy of molecules motion keeps them in random distribution in a solution. The collisions of solute molecules with each other and with solvent (water) molecules impart sufficient energy to exceed the force of gravity. Because molecules in solution are so small, the gravitational force on each individual molecule is insufficient to allow it to settle out.

Question 28

what is a suspension

Answer 28

A suspension is a mixture of insoluble particles in a suspending fluid, in which the particles will eventually settle out due to gravity if allowed to stand for sufficient time. The rate of sedimentation or precipitation depends on the particle density.

Question 29

why is it important to mix a suspension before sampling

Answer 29

When sampling a suspension of cells, it is important to mix the suspension well prior to sampling or you will get different concentrations based on where you sample.

Question 30

how do you express concentration usually

Answer 30

The concentration of a solution is usually expressed in moles per litre. It is the formula weight in grams of a molecule dissolved in one litre of water. Said to be 1 mole / L or 1M.

Question 31

what are typical concentrations of intracellular molecules

Answer 31

Typical concentrations of intracellular molecules are usually less than 1M, and are expressed as mM (millimoles 1 x 10^-3 M), uM (micromoles 1 x 10^-6 M), nM (nanomoles 1 x 10^-9 M), pM (picomoles 1 x 10^-12 M), or even smaller denominations.

Question 32

what are two other ways to express concentration

Answer 32

Concentrations are also sometimes expressed as mass per unit volume, such as mg/L. Another way to express concentration is as a percentage. A v/v percentage is simple the number of parts of solute in 100 parts of the solvent. The parts could also be grams, Kg, Lbs, ounces, etc. A w/v percentage is simply the number of grams in 100 L of solvent.

Question 33

what is dilution

Answer 33

Dilution means adding water of some other fluid to a solution of known concentration, usually called the stock solution. This reduces the concentration in the solution. (Initial Concentration)(Initial Volume) = (Final concentration)(Final volume)

Question 34

how can you express dilutions

Answer 34

a 1/100 dilution means that 1 part of the solute is added to 99 parts of the diluent. The total volume is 100 parts. The expression 1:99 also works to represent this dilution.

Question 35

what is spectrophotometry

Answer 35

Spectrophotometry is a technique used to measure the absorbance of energy, usually in the form of light, by a molecule in solution. Measured using a spectrophotometer.

Question 36

what is the absorbance maximum

Answer 36

The wavelength of light is maximally absorbed by a specific molecule is called the absorbance maximum. For many compounds, absorbance is proportional to the concentration of the absorbing solution and to the length of the light path. This is know as the Beer-Lambert Law

Question 37

what is the Beer-Lambert Law

Answer 37

For many compounds, absorbance is proportional to the concentration of the absorbing solution and to the length of the light path. This is know as the Beer-Lambert Law Absorbance = (Wavelength dependent molar absorptivity coefficient in M^-1cm^-1)(Length of light path in cm)(Concentration of the absorbing solution in M) A = kLC Absorbance has no units

Question 38

what is intensity

Answer 38

It is common to use the energy of the radiation per unit area per unit time, which is called the intensity, I, to measure of the “amount” of electromagnetic radiation impinging on a solution

Question 39

what is transmittance

Answer 39

For a partially transparent sample, the proportion of that intensity that passes through the sample is a measure of the transmittance of the sample. The more transparent, the higher the transmittance. %T = 100 I(t) / I(o) (percent transmittance) = 100 * (Light transmitted through the sample)/(Light falling on the sample)

Question 40

what is absorbance

Answer 40

Since molecules absorb electromagnetic radiation at specific frequencies, we define absorbance of light at wavelength gamma, by a sample in terms of the percent transmittance. Since the amount of radiation absorbed can vary over an extremely wide range, it is useful to define absorbance logarithmically. A = log (100/%T) or A = 2.000 - log(%T) The absorbance of a sample with 100% transmittance will be 0. A = 1 = 10% transmittance A = 2 = 1% transmittance A = 3 = 0.1% transmittance.

Question 41

how do we do spectrophotometry

Answer 41

In spectrophotometry one uses a control solution that contains all the reagents used for the test, except the substance whose concentration is being determined. Thus, there should be no change in absorbance of the control. Any change would mean some factor other than the chromogen of the substance assayed is causing the change in absorbance, and all other absorbances measured with the same chromogen would have to be adjusted for this.

Question 42

what is a blank

Answer 42

It is also necessary to use a blank solution before reading any other absorbance. The blank should consist of everything that is in the test solution, except the chromogen. Sets an absorbance of 0 at a specific wavelength.

Question 43

How do you measure concentration

Answer 43

To measure concentration: 1. Determine the absorption maximum for the substance, set the spectrophotometer to this wavelength 2. Prepare a series of samples of known concentration of the substance, then measure the absorbance of each 3. Plot the values of the absorbance as a function of concentration 4. Verify that absorbance is a linear function 5. Determine the slope of the line of best fit through the experimental points in the absorbance vs concentration graph 6. Use the value of the slope to calculate the concentration of an unknown concentration based on absorbance. (absorbance) = (slope)(concentration of sample)

Question 44

how does the Novaspec Spectrophotometer work

Answer 44

The Novaspec III+ allows the recording of absorbance, transmittance or concentration over the wavelength range 325-1100 nm. Different sizes of cuvettes may be used depending on the nature of volume of solution with which one is working.

Question 45

What are the 8 steps to using the Novaspec III+

Answer 45

1. Turn the instrument on and let calibrate. 2. Select the view options key and press 1 to set parameters. Use left and right arrow keys to select absorbance mode. Press the down arrow key, use the number keypad to enter required wavelength. Press OK key 3. Use kimwipe on outside of cuvette 4. Place the blank Cuvette in Cuvette holder with triangle facing right. Press the SET REFERENCE key (OA) once. The display will read 0.000 5. Remove reagent blank cuvette. 6. Insert the first sample cuvette to be measured, again wiping the outside with cuvette. Select TAKE A MEASUREMENT key are record absorbance 7. If future measurements are to be made at a different wavelength, change the wavelength and then SET REF with the new blank at the new wavelength before measuring sample absorbances. 8. Never leave your last sample in the instrument.