Lecture 3

Basic Biochemistry Experimental Techniques and Skills

Objectives

• To understand the general rule of biochemistry laboratory and receive your lab glassware.

• To be familiar with glassware washing procedure and volumetric pipette operation

Rules for a Safe Lab Environment

• Safety in the biochemistry laboratory involves a cautious attitude and an awareness of potential hazards.

• The number of laboratory accidents can be reduced if every student follows all of the directions given for the experiment and by the instructor.

• Special note should be taken of specific instructions that are given in an experiment to eliminate recognized potential hazards.

Rules for a Safe Lab Environment

• Any breakage of glassware or other breakable laboratory equipment is to be immediately reported to the laboratory instructor.

• Electronic devices, such as cellular phones, and any personal entertainment devices must be turned off or turned to silence mode prior to the beginning of the lab period.

Important roles

• Ware lab coat

• Seat according to your student number

• Clean after the class

Student Responsibility

• PROTECT YOUR EYES.

• When dealing with any biological material or chemical, take all necessary precautions to avoid skin contact

• Wear gloves when working with certain samples and chemicals. Some of the chemicals are toxic or mutagenic.

Standard Laboratory Practices

• Label all samples clearly with your name, date, and contents.

• Gloves are changed whenever they may have become contaminated.

Centrifuge

• For the safety of everybody, please do not exceed the rpm 3000.

Laboratory glassware

• Some of the equipment is now made of plastic for cost and convenience reasons, but glass is still used, because it is relatively inert and transparent.

graduated cylinderstir rod funnel

Laboratory glassware

beaker flask

petri dish microscope slide and cover slip

glass tube

volumetric pipette

Your lab tools

• 18 large test tubes

• 4 small test tubes

• 1 dropper

• 2 beakers (A larger one and a small one)

• 1 rubber ear syringe

• 1 glass rod

Volumetric pipettes

• bulb pipette• allow the user to measure a volu

me of solution extremely accurately.

• These pipettes have a large bulb with a long narrow portion above with a single graduation mark as it is calibrated for a single volume

• Typical volumes are 10, 25, and 50 mL.

Volumetric pipette operation

Ostwald-Folin Pipet

• A special pipette used in measuring viscous fluid such as whole blood.

Graduated pipettes

• consisting of a long tube with a series of graduations

• To learn how to precisely use calibrated variable volume micropipettors and prepare solutions.

• As a scientist, you need to be able to accurately use calibrated variable volume micropipettors to add and transfer small amounts of reagents and samples precisely and accurately. This lab is a chance to test the accuracy of our tools and your understanding of how to appropriately set the micropippetors to precisely deliver the amount of liquid required.

• Glass pipettes are not highly accurate for volumes less than 1 milliliter (1 ml

• Micropipettes are continuously adjustable digital or rotary pipettes. Each pipette can be set to transfer any volume within its own volume range using specially designed tips

Micropipettes

These pipettes operate by piston-driven air displacement.Set ：2.5ul 、 10ul、 20ul、 100ul 、 200ul 、

1000ul and 5000ul

Operation of a micropipette

Do not attempt to set pipet for volumes larger than their maximum, or for volumes less than zero;

doing so will damage the pipet

Operation of a micropipette

• set the volume

significant figures

• All measurements are approximations. A mass measured to 13.2 g is said to have an absolute uncertainty of plus or minus 0.1 g 

• In other words, we are somewhat uncertain about that last digit. A mass of 13.20 g indicates an absolute uncertainty of plus or minus 0.01 g

• The number of significant figures in a result is simply the number of figures that are known with some degree of reliability.

• The number 13.2 is said to have 3 significant figures. The number 13.20 is said to have 4 significant figures.

significant figures• (1) All nonzero digits are significant:

1.234 g has 4 significant figures,1.2 g has 2 significant figures.

• (2) Zeroes between nonzero digits are significant:1002 kg has 4 significant figures,3.07 mL has 3 significant figures.

• (3) Leading zeros to the left of the first nonzero digits are not significant; such zeroes merely indicate the position of the decimal point:0.001 ℃ has only 1 significant figure,0.012 g has 2 significant figures.

• (4) Trailing zeroes that are also to the right of a decimal point in a number are significant:0.0230 mL has 3 significant figures,0.20 g has 2 significant figures.

• (5) When a number ends in zeroes that are not to the right of a decimal point, the zeroes are not necessarily significant:190 miles may be 2 or 3 significant figures,50,600 calories may be 3, 4, or 5 significant figures. The potential ambiguity in the last rule can be avoided by the use of standard exponential, or "scientific," notation. For example, depending on whether the number of significant figures is 3, 4, or 5, we would write 50,600 calories as:5.06 × 104 calories (3 significant figures)5.060 × 104  calories (4 significant figures), or5.0600 × 104  calories (5 significant figures).

significant figures• (1) In addition and subtraction, the result is rounded off to the last co

mmon digit. Another way to state this rule is as follows: in addition and subtraction, the result is rounded off so that it has the same number of digits as the measurement having the fewest decimal places (counting from left to right). For example,100 (assume 3 significant figures) + 23.643 (5 significant figures) = 123.643,which should be rounded to 124 (3 significant figures). Note, however, that it is possible two numbers have no common digits (significant figures in the same digit column).

• (2) In multiplication and division, the result should be rounded off so as to have the same number of significant figures as in the component with the least number of significant figures. For example, 3.0 (2 significant figures ) × 12.60 (4 significant figures) = 37.8000which should be rounded to 38 (2 significant figures).

Operation of a micropipette

• First, set the volume to transfer, then attach the disposable tip, depress the plunger to the first stop, immerse the tip in the sample, slowly draw up the sample with the tip completely immersed, pause for viscous samples, withdraw the tip,

• dispense the sample by pressing the plunger through the first stop to the second stop vertically, withdraw the pipet

• release the tip into the trash

use the appropriate tips

Pipette type Volumes (μL) Tip color

P10 0.5 – 10 white

P20 2 – 20 yellow

P200 20 – 200 yellow

P1000 200 – 1000 blue

• Try depressing the plunger. As the plunger depresses, you will feel a sudden increase in resistance. This is the first “stop”.

• If you continue pushing, you will find a point where the plunger no longer moves downward (the second “stop”).

• When using the pipet, depress the plunger to the first “stop”, place the tip into the liquid, and in a slow, controlled manner, allow the plunger to move upwards. (Do not simply let the plunger go; doing so will cause the liquid to splatter within the tip, resulting in inaccurate volumes and in contamination of the pipet.)

Question

• A P200 Pipette set as shown in the diagram.

• What volume is it set for?

• Is this the proper pipet for this volume?

Precision and Accuracy

• Accuracy is a measure of proximity to the true value or the expected value for a measurement.

• Precision is a measure of reproducibility.

Manual MixingManual mixing is a quick method to prepare a homogeneous mixture.  Some examples of manual mixing techniques are included below.

• Gently Striking the Side of the Container: Small volumes in a test test tube can be mixed by gently striking the side of the test tube with a finger 

• Shaking the Container: A closed container can be shaken only if no gas will be formed. For a test tube that is less than half full, you can mix effectively by rapidly shaking the bottom of the tube left and right while keeping the top in one place. NEVER use your finger or hand to seal a container that will be shaken.

• Swirling the Container: To swirl the container, lift the container and move the base of the container in small circles.  Generally, it is not a good practice to swirl solutions in wide-mouthed container such as a beaker, since splashing might occur.

• Stirring with a Glass Rod: Glass stirring rods can be used to mix a heterogeneous mixture.

Washing

• When washing, soap and detergent may be used.

• When you use a brush try not to scratch the glass surface.

• Insert the brush by twist it

• Rinse with water

Rinsing

• It is imperative that all soap, detergents and other cleaning fluids be removed from glassware before use.

• Slight traces of which will interfere with serologic and cultural reactions.

• After cleaning, rinse the glassware with running water. When test tubes, graduates, flasks and similar containers are rinsed with water, allow the water to run into and over them for a short time, then partly fill each piece with water, thoroughly shake and empty at least six times.

• Rinse with distilled water for 2-3 times.• Put the tubes up side down untill they are dry

Clean with chromic acid

• If glassware becomes clouded or dirty or contains coagulated organic matter, it must be cleansed with chromic acid cleaning solution

• Chromic acid is a mixture made by adding concentrated sulfuric acid to a dichromate (Cr2O72−)

• The dichromate should be handled with extreme care because it is a powerful corrosive and carcinogen

Lab report

• The title of an experiment

• The aim of an experiment

• The principle of an experiment

• The reagents and equipments

• The experimental procedure

• Results

• Answers to the assignment questions

Please turned in the lab report in the next class

Experiments for today

• Recognize the different type of pipettes and decide the significant figures when using a graduated pipette or a micropipette

• use 0.5mL, 1mL, 2mL, 5mL and 10mL volumetric pipette to transfer the solution of 0.25mL, 0.8mL, 1.4mL, 3.8mL and 8.5mL to 5 tubes and choose a proper way to mix them

• Use 20μL, 200 μL and 1000 μL micropipette to transfer the solution of 12.8 μL, 145 μL and 850μL into 3 tubes and mix them properly

Assignment Questions

• Which of the following is a better way to rinse a tube? Why? A. Rinse once with 2 ml distilled water. B. Rinse twice and use 1 ml distilled water each time

• What is significant figures? How many significant figures for the following numbers. 123.0, 123, 0.123, 0.1230

• The smallest unit of a 1mL volumetric pipette is 0.01 ml. How many digits after the decimal point when you read the measurement?