Chapter X: Stock Solution Preparation
1. Introduction
Stock solutions are concentrated forms of reagents that serve as the basis for preparing
working solutions in laboratory experiments. They ensure consistency, accuracy, and ease
of use in biochemical and molecular biology procedures. This chapter details the
preparation of essential stock solutions, including Tris-HCl, EDTA, NaOH, NaCl, and TAE
buffer, which were used in this research.
2. Materials and Methods
2.1 1M Tris-HCl (pH 8.0)
Reagents:
• Tris base: 60.75g
• Distilled Water (dH₂O): 400mL
• Concentrated HCl (for pH adjustment)
Procedure:
1. Weighed 60.75g of Tris base and dissolved it in 400mL of dH₂O.
2. Adjusted the pH to 8.0 using concentrated HCl.
3. Brought the final volume to 500mL with dH₂O.
4. Stored at room temperature.
2.2 0.5M EDTA (pH 8.0)
Reagents:
• Na₂EDTA.2H₂O: 93.05g
• dH₂O: 350mL
• 10M NaOH (for pH adjustment)
Procedure:
1. Weighed 93.05g of Na₂EDTA.2H₂O and dissolved it in 350mL of dH₂O.
2. Adjusted the pH to 8.0 using 10M NaOH.
3. Added dH₂O to make a final volume of 500mL.
4. Stored at room temperature.
2.3 10M NaOH
Reagents:
• NaOH pellets: 200g
• dH₂O to 500mL
Procedure:
1. Weighed 200g of NaOH pellets.
2. Slowly added NaOH to dH₂O with continuous stirring.
3. Adjusted the final volume to 500mL.
4. Stored at room temperature.
2.4 6M NaCl
Reagents:
• NaCl: 175.5g
• dH₂O: up to 500mL
Procedure:
1. Weighed 175.5g of NaCl and dissolved it in dH₂O using a magnetic stirrer.
2. Brought the final volume to 500mL.
3. Stored at room temperature.
2.5 50X TAE Buffer (Tris-Acetate-EDTA)
Reagents:
• Tris base: 121g
• Glacial Acetic Acid: 28.5mL
• 0.5M EDTA (pH 8.0): 50mL
• dH₂O: up to 500mL
Procedure:
1. Weighed 121g of Tris base and dissolved it in dH₂O.
2. Added 28.5mL of glacial acetic acid.
3. Added 50mL of 0.5M EDTA (pH 8.0).
4. Adjusted the final volume to 500mL.
5. Stored at room temperature.
3. Working Solution Preparation
3.1 TE Buffer (10mM Tris, 2mM EDTA, pH 8.0)
Reagents:
• 1M Tris-HCl (pH 8.0): 5mL
• 0.5M EDTA (pH 8.0): 0.2mL
• dH₂O: up to 500mL
Procedure:
1. Mixed 5mL of 1M Tris-HCl and 0.2mL of 0.5M EDTA in dH₂O.
2. Adjusted the final volume to 500mL.
3. Stored at room temperature.
3.2 Proteinase K (20mg/mL)
Reagents:
• Proteinase K: 100mg
• Ultra-pure dH₂O: 5mL
Procedure:
1. Dissolved 100mg of lyophilized Proteinase K in 5mL of ultra-pure dH₂O.
2. Aliquoted and stored at -20°C.
3.3 TEN Buffer (10mM Tris, 2mM EDTA, 400mM NaCl)
Reagents:
• 1M Tris-HCl (pH 8.0): 5mL
• 5M NaCl: 40mL
• 0.5M EDTA: 2mL
• dH₂O: up to 500mL
Procedure:
1. Mixed the reagents in dH₂O.
2. Adjusted the final volume to 500mL.
3. Stored at room temperature.
3.4 SDS 10% (w/v)
Reagents:
• Sodium Dodecyl Sulfate (SDS): 10g
• dH₂O: 400mL
Procedure:
1. Dissolved 10g of SDS in 400mL of dH₂O.
2. Heated the solution to approximately 65°C to dissolve SDS completely.
3. Adjusted the final volume to 500mL with ultra-pure dH₂O.
4. Stored at room temperature.
4. Conclusion
The stock and working solutions described in this chapter were essential for molecular
biology experiments. Their precise preparation ensured experimental accuracy,
reproducibility, and efficiency in downstream applications.
