LAB SESSION #2 - An introduction to Biochemistry laboratory and laboratory techniques.

1. Syllabus
2. Pipetting error - Lab  bulletins from Artel-USA
               pipetting technique and error - ten tips sources of error effects of poor technique
               accuracy vs. precision - accuracy and precision

3. The laboratory notebook
4. Robyt and White text: Chapter 1- Analyzing and Reporting Experimental Data; Chapter 2 - 2.1-2.4

Procedure Outline -
You should meet with your partner and prepare a procedure for this experiment.
In discussion with your partner(s), design an experiment to test the accuracy of a micropipettor
You will also compare the 1000 ul pipet to the accuracy of a standard 1ml glass pipet. You must determine how you will check the calibration of both pipets.

Things to consider in your experimental design:
**How many replicates will be needed in order to obtain reasonable statistical data?
**What variables could affect pipetting accuracy?
**The micropipettes can be used for a range of volume measurement and delivery; how can the instrument's calibration be tested over this entire range?
 ** Over the course of the semester you will be measuring volumes from 5 to 1000 microliters. What pipets will you calibrate?

Information on the correct usage of the micropipettors is provided below.  Please familiarize yourself with this material.

YOU MUST PREPARE THIS PROCEDURE BEFORE THE LABORATORY PERIOD.  FAILURE TO PREPARE A PROCEDURE WILL RESULT IN A GRADE OF 0 FOR THE LABORATORY.

1. Introduction to biochemistry lab, policies, procedures, etc. (review syllabus)

2. An exercise illustrating:
a) experimental design
b) calibration and use of micropipettors
c) precision and accuracy
d) statistical analysis of data

The Use of Micropippetors: You will calibrate and determine the accuracy and precision of your micropipettors and a glass pipet. A written summary of your results should accompany the table or graph that you prepare from your data (see post lab).

This will be a relatively simple exercise with general objectives:
a) to think about what goes into designing an experiment;
b) to introduce the use of micro-pipettes;
c) to review the concepts of accuracy, precision and statistical analysis of data;
d) to use standard computer software (e.g. spread sheets and simple plotting programs) for data analysis.

It is imperative that biochemists be able to measure small volumes (1 to 1000 microliters or 10^-6 L) of liquids. Successful experiments depend both on the accuracy of measuring and delivering such small volumes and on the researcher's ability to use the pipets correctly and consistently. In this laboratory, you will learn to correctly utilize and calibrate micropipettors for use in experiments throughout the semester. Information on these instruments from the company manual are available in the laboratory.

**Carry out your experimental procedure after you check your logic with me.
**Data collected should be entered directly into your laboratory notebooks.
**Use your data to determine the accuracy and precision of your micro pipet. Compare this to the glass pipet.

1. What factors can affect pipet performance (i.e. accuracy and precision)?
2.  How did you resolve the issue of accuracy for a variable range pipet?
3.  Comment on the accuracy and precision of your measurements. (This could be done in sentence form or graphically)
4.  What is more accurate at a given volume, a glass pipet or the pipettor?  What is more precise?  Does your answer vary with selected volume?
5.  Are smaller volumes measured with more or less accuracy with the pipettor?  What about precision?
6.  Based on your data, if it was critical that you obtain exactly a 1.00 mL volume of solution, which instrument would you use?
7.  Did you notice any changes in accuracy and precision associated with operator changes or operator "practice"?

You will analyze the data collected about the accuracy and precision of the micropipettor and the glass pipet that you calibrated. Enter your collected data into a computer spreadsheet. Use the spreadsheet tools to do the data analysis and create tables or graphs that best illustrate your results. Determine the mean and standard deviation of your data. A written summary of your results should accompany the table or graph that you prepare from your data.

The answers to the post lab questions and post lab data will be collected with the prelab for week 2. These questions should be answered in your notebook. Carbon pages will be collected at the beginning of the period.  Your grade for this lab will be based upon the prelab and the collected post lab.

Instructions are provided for the Bio- Rad Pippettors - other pipettors operate in kind

INTRODUCTION
Bio-Rad pipettors are available in standard volumes from 0.5 ml to 100 ml.
Bio-Rad instruments are equipped with an adjustable tip ejector eliminating the risk of contamination.
Bio-Rad instruments are made of corrosion resistant materials and require minimal preventative maintenance.
Bio-Rad instruments accept white, yellow or blue tips; color of the correct tip is indicated by the color of the push button.

VOLUME CHANGE
These continuously adjustable pipettors are remarkably free from mechanical backlash. However, it is considered good practice to slightly over-set the actual volume required. This technique ensures that the best accuracy is obtained. Select the correct pipettor for the operating range required. Using the knurled nut in the handle adjust to the desired volume using the technique previously described. The volume set is displayed in the window in the center of the pipettor body.
 

SUGGESTED METHOD OF USE
a) Select the correct tip and place it firmly on the nose of the pipettor using a slight screwing action. NEVER USE THE PIPETTOR WITHOUT A TIP ATTACHED as serious damage will result.
b) Prior to the immersion of the tip of the fluid under test, depress the button to the first stop.
c) With the pipettor held vertically immerse the tip to a depth of 3-5 mm in the fluid under test.
d) Release the button in a controlled manner to aspirate the volume selected.
e) Remove the tip slowly from the fluid holding the pipettor vertically. DO NOT WIPE THE TIP with a tissue; this tends to wick martial from the pipet. Touching briefly to the side of the rim of the container will take off most drops  found on the tip.

Bio-Rad Models (We have two sets of these) (Blue with different colored tops for the different sizes; instruction manual in the laboratory)
Top color:
White - Used for volumes between 0.5 to 10 uL only
Bright yellow - Used for volumes between 2 to 20  uL only
Golden yellow- Used for volumes between 20 to 200  uL only
Blue - Used for volumes between 100 to 1000  uL only

Gilson Models ("Pipetmen", blue)
P-20 - Used for volumes between 1 - 20  uL only
P-200 - Used for volumes between 20 - 200  uL only
P-1000 - Used for volumes between 200 - 1000  uL (1 mL) only

Fisher Brand Models
(Black with different colored tops for the different sizes)
Top color:
Green - Used for volumes between 10 - 50  uL only
Red - Used for volumes between 40 - 200  uL only
Blue - Used for volumes between 200 - 1000  uL (1 mL) only

Casually all many people will us the term P-x   x being the top volume for the pipet in question for all brands of pipets.

**ALWAYS use plastic tips with these pipettors

All three brands of pipettors that deliver the larger volumes (either 100 ­ 1000 or 200 ­ 1000 L) utilize the same tips.

Larger relative volumes will cavitate and splash if the plunger is released to quickly. always move the plunger slowly

The smaller pipettors use different tips depending on the model.

The smallest Bio-Rad pipettors (0.5 to 10 L) use a special type of tip. For this class, we will rarely use this size of pipettor. You do not have to calibrate this pipettor.

PIPET CARE - PAY ATTENTION TO THESE DETAILS!
**Always Place the pipet into a stand when you are not using it 

**NEVER Lay a pipet on the bench top with a tip on it

**NEVER adjust pipet settings to values above or below the range indicated on the pipettor. This will ruin the calibration and possibly destroy the instrument.

**NEVER force any part on the pipet

**Keep the pipets CLEAN.

**If you are not sure how to use the pipets, ASK!