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Performing dilutions - Calculate concentration of substance in dilutions.

Protocol Assignment

Principle:  To write a set of directions that clearly and fully describes the steps that are required for a laboratory scientist to perform dilutions and calculate the concentration of substance in those dilutions.

Background:  You have followed a protocol for each and every laboratory exercise throughout the semester.  As a professional, you may be required to write the protocols of your department such that your colleagues can perform a task in an exact and precise manner. 

You must now use the information you’ve learned on how to write a protocol (first day of lecture) and the skills you’ve acquired in the last several weeks regarding dilutions and pipetting, to direct a classmate in a laboratory exercise.

Materials/Equipment:

1.    Deionized water

2.    Stock Cibracon blue dye (20 mg/dL)

3.    Spectrophotometer

4.    Micropipettes (2-20uL, 20-200uL,and 100-1000 uL)

5.    Serological pipettes (2ml, 5ml and 10 mL)

6.    Kim wipes

7.    Parafilm

8.    Tubes

Method: 

1.    Write a formal protocol that includes all the necessary components (title, principle, background, etc.) and that instructs a classmate in how to make dilutions.  You must instruct the student to make several single dilutions and one serial dilution. Assume that the student knows NOTHING with regard to dilutions but has a basic knowledge of pipetting.

A.    Your protocol must provide ALL the steps to make various dilutions and include:

                      i.        What dilution is being made

                     ii.        The pipettes that must be used in order to make the dilution

                    iii.        The volumes of stock solution and water that must be used to make that dilution

·         You will need to instruct the student to make the dilutions using a volume that you choose, in the range of 2 mL - 5 mL.  You do not need to have the same volume for all tubes.

·         The minimum volume needed for the spectrophotometer is 2 mL and the maximum volume that the tubes can hold is 5 mL. 

                   iv.        The directions for determining the absorbance by spectrophotometry

                     v.        A space for the student to record spectrophotometric absorbance readings and to calculate the concentration of each dilution

·         EACH tube in the individual dilutions will be analyzed, but ONLY the last tube in the serial dilution will be analyzed

·         Don’t forget to instruct the student to determine all the components that are required to calculate concentration (Remember Beer’s Law!!)

B.    Provide a space at the top of the protocol for “Written by:” and “Completed by:”

C.   A 20 mg/dL stock solution will be used to make the following dilutions:

1.      1/3

2.      1/6

3.      1/10

4.      1/15

5.      1/25

6.      A serial, 5-part dilution, in which each dilution is a 1/2 dilution

Performing dilutions - Calculate concentration of substance in dilutions.

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