Lab #01: Biochemistry Basics

Question 1

Top Loading Single Pan Balances

Answer 1

Most popular type of balance for general laboratory use.

Question 2

Double Pan Balances

Answer 2

Used to equilibrate volume or weight, especially before centrifugation.

Question 3

Analytical Balance

Answer 3

Used to obtai extremely accurate measurements of mass. Operating range is 0.0001 to 99 grams.

Question 4

Rules For Using the Balance

Answer 4

1. Always weigh material into a container. Do not put chemicals directly on balance tray.
2. Check chemical labels for any hazard warnings.
3. Always use a clean spatula to remove solid chemicals from stock bottles.
4. Cover chemicals tightly when done.
5. Clean up any spills.

Question 5

Graduated Cylinders

Answer 5

· Volume is measured in liters, milliliters, and microliters. Measurements of liquids and solutions from 1 mL to several liters is accomplished through use of graduated cyliners. When using one, line the bottom of the meniscus up with the calibration line on the cylinder.

Question 6

Pipets: Volumetric

Answer 6

Volumetric – calibrated to deliver one exact volume. These have capacities of 0.5mL to 20mL.

Question 7

Pipets: Mohr

Answer 7

Mohr – calibrated to deliver any # on the graduated scale which ends on the straight tube above the beginning of the tapered end.

Question 8

Pipets: Serological

Answer 8

Serological – calibrated to deliver any # on the graduated scale; its graduated to the tip. Serological pipets are made to be blown out unless specifications of how to deliver are noted on them. When moving volumes to a test tube, the tip of the pipet should be near the bottom of the tube. The tip should be touched to the side of the container at the end of the measurement to remove the last drop in the pipet.

Question 9

Identifying Serological Pipets

Answer 9

Can be done by the label near the top of the pipet, which identifies capacity and the volume gradations.

For example, a pipet labeled “5 x 0.1 mL” will hold 5 mL, and the volume is marked off in 0.1 mL increments.

They’re reuseable, when you’re done with one, place it carefully in the “Used Pipet” container with tip down.

Question 10

Use of a Pi-Pump

Answer 10

· Use of a Pi-Pump

1. The GREEN pi-pump is for 5 and 10 mL pipets.
2. The BLUE pi-pump is for 1 and 2 mL pipets.
3. Carefully inset the pipet into the pi-pump
4. Holding the pi-pump with one hand, insert the pipet into the solution.
5. Roll wheel up. Watch level of solution, careful to not draw it up in the pi-pump.
6. To empty pipet, hold it over the container or tube. Push the lever until the meniscus is at the desired level. Roll the wheel down to get the last bit of liquid out of a serological pipet.
7. Remove pipet and place in container.
8. Return pi-pump to the bench in the lab.

Question 11

Pasteur Pipets

Answer 11

· Pasteur pipets are small disposable glass pipets used to add/remove small unmeasured # of liquid. Max 2mL and are used with a rubber Pasteur pipet bulb.

Question 12

Transfer Pipets

Answer 12

Disposable pipets used in same manner as Pasteur pipets. They are made of soft plastic and come with a bulb molded in as part of the pipet.

Question 13

Micropipettors

Answer 13

· Micropipettors are designed to deliver volumes from 0.5 to 1000 microliters. Used with disposable plastic tips. Some models measure only a single fixed volume, but you will be using pipettors that are adjustable through a certain range of volumes.

Question 14

Use of Micropipettors

Answer 14

· Use of Micropipettors

1. The min and max volumes are displayed on the top of the pipettor. Do not use a pipettor outside of the indicated range, or you’ll measure inaccurately and may break the pipettor.
2. Set desired volume by unlocking black ring near top of pipettor. Turn knob at top of plunger. Once volume is set, retighten the black locking ring.
3. Attach plastic tip to shaft of pipettor. Press on firmly with a slight twisting motion to ensure an airtight seal.
4. Depress plunger a few times to become familiar with two “stop” positions. Then depress plunger to first stop to measure set volume. Insert the pipet tip in liquid and slowly let up plunger. If release plunger all at once, the liquid will boil up into the pipettor and will require special cleaning.
5. Wait a few sec to make sure tip is full. Make sure to keep pipettor within 45 degrees of vertical during filling and expelling liquid.
6. Withdraw tip from the liquid and touch it to side of vessel to drain any liquid from outside of tip.
7. To transfer measured volume of liquid to another vessel, place tip against the side of the vessel, depress the plunger to the first stop, wait 1-2 sec, and depress to the second stop. All liquid should be expelled from the tip.
8. Keeping plunger depressed, remove pipettor from vessel. Use ejector button to discard the tip.

Question 15

Aliquot

Answer 15

· An aliquot refers to a small sample taken from a larger volume. They’re often taken at each step of a protocol and used to analyze success of protocol. Also may be used as a verb.

Question 16

When is a measurement accurate?

Answer 16

· A measurement is accurate if it measures the true value.

Question 17

Precision

Answer 17

· Precision refers to the reproducibility of measurement.

Question 18

Accuracy/Precision & Device

Answer 18

· Accuracy and precision are determined by the type of device used for measurement as well as the technique used for making it. One of most important skills to learn is choosing appropriate device for making a particular measurement, and knowing which measurements need to be done with high accuracy.

Question 19

Normal Solution

Answer 19

· Normal Solution - 1 normal solution (1N) has one equivalent of a substance per liter of solution. Used for acids and bases, where an equivalent is defined as weight of a substance that’ll combine with 1 mole of H+ or OH- ions. For example, 1 mole of HCl contains 1 mole of ionizable H+, which would be able to combine with 1 mole of OH- ions. This means that a 1N solution of HCl is equivalent to a 1M solution. Sulfuric acid contains 2 moles of H+ per mole of itself so a 1M solution is equivalent to a 2N solution.

Question 20

How Else Solutions Can Be Described Without Use of Molarity or Normality

Answer 20

· Other solutions are described by # of a substance dissolved in a particular volume, without regard to the number of moles or equivalents involved. Solutions of proteins and DNA molecules are generally described in terms of milligrams or micrograms per mL. Other times solution might be described as containing a certain # of light-absorbing material per mL. For ex, a solution of protein that gives an absorbance of 0.42 when measured in a spectrophotometer at a wavelength of 280nm could be described as having a concentration of 0.42 A280 units/mL.

Question 21

Solutions Described In Terms of %

Answer 21

· Solutions also described in terms of %. In biochemistry labs, a 50% (w/v) solutions has 50 g of a substance dissolved in a final volume of 100 mL. This is referred to as weight/volume solution, cause percentage refers to weight of a solid substance that is dissolved in a 100mL final volume. % solutions made by mixing another liquid with H2o r volume/volume solutions. For ex., 70% ethanol contains 70 mL of ethanol mixed with enough water to make a final volume of 100mL.

Question 22

Water Part of Molecular Weight?

Answer 22

· Some compounds bind water molecules in an unspecified way when they crystallize, and this water is included in calculation of formula weight. Many compounds have FW out there with or without this water.

Question 23

Dilution

Answer 23

A dilution is made by addition of more solvent to a solution, thus decreasing the concentration. Think of making orange juice from frozen concreate: the concentrated juice is diluted by addition of water. If we use 3 cans of water to 1 can of concrete, the final concentration of juice will be 1/4 of the original concentration beause the final volume is four times the original volume.

Question 24

C1V1 = C2V2

Answer 24

· C1V1 = C2V2
• C1 = original (stock) concentration
• V1 = volume of stock needed
• C2 = final concentration desired
• V2 = final volume of solution to be made.

Question 25

Fold Dilutions From a Concentrated Stock

Answer 25

Other dilutions are defined by ratio of the initial and final concentrations. For a volume to volume dilution (v/v), this is also the ratio of the initial to final volumes! For example, in the orange juice dilution example, a 4 fold v/v dilution was made. It’s important to remember that this represents 1 volume of the original solution in a final volume that is four times as great. Started with 1 can of orange juice concrete, added 3 cans of water, and finished with 4 cans of juice. A 4-fold dilution.

For a 2-fold dilution, 1/2 of the final volume, or 5 mL, will be the stock solution.
For a 5-fold dilution, 1/5 of the final volume, or 2 mL, will be the stock solution.
For a 10-fold dilution, 1/10 of the final volume, or 1 mL, will be stock solution.

Question 26

Serial Dilution

Answer 26

· Serial dilution is where a series of identical dilutions are made in order to reduce the concentration to the desired level. Used for solutions that need to be extensively diluted. Using C1V1 = C2V2, you determine that you’d have to measure 1 microliter of 10M HCl to make this solution in usual way. You’d have to be very confident in accuracy of micropipettor to do it this way. A safer way is to make a series of dilutions that’ll allow you to measure larger volumes and gradually reduce the concentration.

For a serial 10-fold dilution, you would do a series of 10-fold dilutions as follows:

Total Dilution - Volume of Stock - Volume of Water - Final Concentration
10 Fold - 1mL of 10M HCl - 9mL - 1M HCl
100 Fold - 1mL of 1M HCl - 9mL - 0.1 HCl
1000 Fold - 1mL of 0.1M HCl - 9mL - 0.01M HCl
10,000 Fold - 1mL of 0.01MHCl - 9mL - 0.001M HCl