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Stock Solutions and Media Preparation

The document outlines the preparation of various stock solutions and culture media essential for molecular biology experiments, including Tris-HCl, EDTA, sodium acetate, Luria Agar, and Luria Bertani. It explains key concepts such as moles, molarity, stock solutions, and dilution formulas, as well as the importance of sterilization techniques. The procedure details the specific amounts and pH adjustments required for each solution, confirming successful preparation of all five stock solutions.

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banzemaria19
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9 views5 pages

Stock Solutions and Media Preparation

The document outlines the preparation of various stock solutions and culture media essential for molecular biology experiments, including Tris-HCl, EDTA, sodium acetate, Luria Agar, and Luria Bertani. It explains key concepts such as moles, molarity, stock solutions, and dilution formulas, as well as the importance of sterilization techniques. The procedure details the specific amounts and pH adjustments required for each solution, confirming successful preparation of all five stock solutions.

Uploaded by

banzemaria19
Copyright
© All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd

Aim

To calculate and prepare various stock solutions and culture media used in molecular biology
experiments.

Material required

Tris-HCl
EDTA
Sodium acetate
Luria Agar
Luria Bertani (LB) powder
Distilled water
Measuring cylinder
Beakers
Balance
pH meter
Autoclave
Laminar air flow

Theory
1. Mole
A mole is the amount of a substance that contains 6.022 × 10²³ particles (Avogadro’s number).
2. Molarity (M)
Molarity is defined as the number of moles of solute per liter of solution.

Molarity M= Moles of solute/ Volume of Solution in liters

3. Stock Solution

A stock solution is a highly concentrated solution from which diluted (working)


solutions are prepared.

4. Working Solution

A working solution is a diluted solution prepared from a stock solution and used
directly in experiments.

5. Dilution Formula

C1V1 = C2V2

Where:
C1 = concentration of stock solution
V1 = volume of stock solution
C2 = concentration of working solution
V2 = final volume of working solution

Culture media

Culture media are substances used to grow microorganisms in the laboratory.


Types of culture media:

Simple media
Enriched media
Enrichment media

Selective media
Differential media
Transport media

Sterilization Technique

Sterilization is the process of eliminating all microorganisms, including spores.


In this experiment:
Autoclave was used for sterilizing media and solutions using steam under
pressure.
Laminar air flow was used to maintain aseptic conditions during preparation and
handling of solutions.
8. Importance / Application of the Experiment
●​ This experiment is useful because:
●​ Prepared solutions are used for bacterial culture such as E. coli.
●​ Buffers like Tris-HCl and EDTA are used in DNA extraction and storage.
●​ Sodium acetate is used in DNA precipitation.
●​ Luria Agar and LB media are used for growing and maintaining bacterial
colonies in molecular biology experiments.

Procedure:

The following solutions and media were prepared:

Tris-HCl (100 ml & 200 ml, 1 M, pH = 8–8.8)


EDTA (100 ml, 0.5 M, pH = 8)
Sodium acetate (50 ml, 3 M, pH = 8–8.8)
Luria Agar (200 ml)
Luria Bertani (210 ml)

1.​ Tris-HCl

Volume = 100 ml, 200 ml


Molarity = 1 M
Molecular mass of Tris-HCl = 121.14 g/mol

1 = x × 1000/ 121.14×100

Therefore, 12.114 g of Tris-HCl was added to 100 ml water to prepare 1 M solution.


Similarly, 24.228 g was added to 200 ml water.
pH was adjusted to 8–8.8 using a pH meter.

2.​ EDTA
Volume = 100 ml
Molarity = 0.5 M
Molecular mass of EDTA = 292.24 g/mol
x = 14.612 g
Therefore, 14.612 g of EDTA was added to 100 ml water and pH was adjusted to
8–8.8.

3. Sodium Acetate
Volume = 50 ml
Molarity = 3 M
Molecular mass = 136.08 g/mol
x = 20.412 g
Therefore, 20.412 g of sodium acetate was added to 50 ml water and pH adjusted
to 8–8.8

4. Luria Agar
35 g in 1000 ml water
For 200 ml:
Therefore, 7 g of Luria Agar was added to 200 ml water.

5. Luria Bertani
20 g in 1000 ml water
For 210 ml:
Therefore, 4.2 g of LB powder was added to 210 ml water.

LA LB

Results:

All five stock solutions were successfully prepared after performing the necessary
calculations.

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