Dilution, pipetting and spectrophotometer

BIOL368

Genetics and Cell Biology Laboratory

Project Number: 1

Project title: Dilution, Pipetting and Spectrophotometer

Name: Syeda Irine Islam        

Student ID: 26596444

Lab section: 02

Group:7

Lab partner: Shirley Tan

Date submitted: Wednesday, 21st September 2016

Introduction

In this experiment, three common practices in science laboratories will be reviewed: dilution, pipetting and spectrophotometry.

Pipetting is a critical skill in molecular biology laboratories as well as in other science fields^[1]. A pipette is an instrument that allows to accurately measure and dispense very small volumes. Micropipettes allow to measure volumes in microliters (μL). They can vary in range: P10 micropipettes  allows to measure volumes from 0.5 μL to 10 μL, for P20 the range is from 2 to 20 μL, 20-200μL for P200 and 100-1000 μL for P1000^[2]. Improper use of micropipettes leads to errors. Therefore, experimenters should learn which tip to use depending of the size of the micropipette, how to handle the plunger button smoothly, how to adjust the volume and how to properly hold it. The first purpose of this experiment is to be able to properly use micropipettes of different range by practicing with distilled water. Since the density of water is 1 kg/L, its volume should be equivalent to its weight. Thus, the accuracy of the pipetting skills will be tested by pipetting given amounts of water and measuring their weight on an electrical balance.

When a solution is too concentrated, it is useful to dilute it; in other words, decrease its concentration. Dilution is a common technique in laboratories, as the stock solutions that are purchased are at very high concentrations^[3]. One can obtain the desired concentration from a stock solution by different methods. Knowing the dilution factor of the final solution can be useful, as it provides the ratio of the initial and final volume^[4]. A simple dilution requires to take a certain amount of the stock solution and to mix it with the required amount of solvent to obtain the desired concentration. In a serial dilution, the stock solution is diluted multiple time in a row^[5]. As a result, the dilution factor remains constant and the concentration decreases exponentially^[6].  The final dilution factor is obtained by multiplying the dilution factor of each dilution in the series. Being able to calculate volumes and concentrations from given data is an essential skill for performing dilutions. Therefore, the second purpose of this lab is to be able to dilute a Coomassie Blue stock solution to the desired concentrations. This part will also help ensure that the pipetting skills learned previously are accurate, as small scaled dilutions will be performed.

Spectrophotometers are instruments that measure the  

Materials and Methods

The materials and methods used in this experiment are the same as in the Biology 368 Lab Manual^[7].

Figures and Tables

Part A. Pipetting exercise

Table 1: Weight and percentage error data obtained by each partner in distilled water pipetting exercise

Calculations:

• Expected weight = volume of water x density of water

Example with 56.5 μL:
Convert density of 1kg/L to mg/μL: 1 kg/L x  x  = 1 mg/μL
56.5 μL x 1 mg/μL[pic 1][pic 2]

• % Error =   x 100[pic 3]

example with 812 µl (Syeda)

 x 100 = 0.91133 %= 0.911%[pic 4]

• Average =   [pic 5]

Example with average at P200 for weight observed:   = 57.0 mg[pic 6]

Part B. Spectrophotometer exercise

Table 2:  Volumes of 50% ethanol and Coomassie Blue stock solution (200 µg/mL) used to prepare diluted samples of various concentrations and their absorbance values at λ=540 nm.

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