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Step-by-Step Guide to Preparing Common Laboratory Solutions, Buffers and Culture Media

The variety of solutions commonly used in laboratories is vast, and the preparation methods are diverse. Are you overwhelmed by these complexities? Today, MCE has compiled the preparation methods for nearly thirty commonly used solutions, buffers, and culture media for you. This covers various fields such as molecular biology, protein analysis, cell culture, and microbial culture.

1. Solution I, II, III for plasmid extraction

Alkaline lysis is a commonly used method for extracting plasmid DNA and is widely applied. Solution I can chelate divalent metal ions to inhibit the activity of DNase. Solution II aims to dissolve the cells, thus releasing the DNA. Solution III serves to precipitate proteins and neutralize the reaction.

Type of Solution System Formula
Solution I 25 mM Tris-HCl ( pH 8.0 )
10 mM EDTA
50 mM Glucose		 • Take 25 mL of 1 N Tris-HCl solution (pH 8.0), along with 20 mL of 0.5 M EDTA (pH 8.0), and 45 mL of 20% Glucose. Then place these into a 1 L beaker.
• Add 910 mL of ultrapure water and mix thoroughly.
• Up to 1 L, sterilize at high temperature and high pressure, and store at 4℃.
Solution II 200 mM NaOH
1% (w/v) SDS		 • Measure 50 mL of 10% SDS solution and 50 mL of 2 N NaOH into a 500 mL beaker;
• Up to 500 mL with ultrapure water, and mix thoroughly.
Note:
(1) Store at room temperature, preferably no more than one month;
(2) During the preparation process, SDS is prone to bubbles, so do not stir vigorously.
Solution III 3 M KOAc
5 M CH2COOH		 • Measure 147 g KOAc and 57.5 mL CH2COOH into a 500 mL beaker.
• Add 300 mL ultrapure water and mix thoroughly.
• Up to 500 mL with ultrapure water.
• After high temperature and high pressure sterilization, store at 4℃.
2、TBE, TAE and MOPS buffers for nucleic acid electrophoresis
TBE, TAE, and MOPS are among the most common buffers stored in laboratories. However, their buffering capacities and application scenarios do have some differences. The buffering capacity of TAE is weaker than that of TBE. Additionally, TBE offers a higher resolution when running gels compared to TAE. In practice, TBE is commonly used for electrophoretic analysis of small DNA fragments. Meanwhile, TAE is suitable for analyses involving large DNA fragments and subsequent enzymatic experiments. MOPS is primarily used for RNA electrophoresis.
Type of Solution System Formula
TAE Buffer ( 50X ) 2 M Tris-acetic acid
100 mM EDTA
pH 8.0		 • 242 g Tris, dissolved in ultrapure water.
• Add 57.1 mL of glacial acetic acid and 100 mL of 0.5 M EDTA solution (pH 8.0). After mixing, adjust up to 1L.
TBE Buffer ( 10X ) 890 mM Tris-Boric acid
20 mM EDTA
pH 8.0		 • 108 g Tris and 55 g boric acid were dissolved in 900 mL ultrapure water.
• Add 40 mL of 0.5 M EDTA solution (pH 8.0). After mixing, adjust up to 1L.
MOPS Buffer ( 10X ) 200 mM MOPS
20 mM NaOAc
10 mM EDTA		 • Add 41.8 g of MOPS to 800 mL of DEPC water. Then mix until dissolved.
• Adjust to pH 7.0 with 2 N NaOH.
• Add 20 mL of 1 M NaOAc ( DEPC treated) and 20 mL of 0.5 M EDTA (pH 8.0) (DEPC treated).
• Up to 1 L with DEPC-treated water . Remove impurities using a 0.45 µm filter and store at room temperature away from light.
Note：The solution can still be used if it turns yellow. However, if it turns black, avoid using it.
3、PAGE buffer

There is a vast variety of buffers for protein electrophoresis. The choice depends on the gel system you use. If the gel system is Tris-glycine, opt for Tris-glycine buffer. For a Bis-Tris system, choose either MES or MOPS buffer. When using a Tris-tricine system, select Tricine buffer.

PAGE buffer can be divided into two categories: denaturing and non-denaturing. The key difference is that denaturing buffers require the addition of SDS to neutralize charges. Denaturing buffers are typically used in WB and IP studies, where the primary focus is determining molecular weights. On the other hand, non-denaturing buffers are frequently utilized when studying protein spatial structures and isoelectric points.

Type of Solution System Formula
Tris-Glycine-SDS ( 10X ) 25 mM Tris Base
192 mM glycine
0.1 % SDS
pH 8.3		 • 29 g Tris, 10 g SDS, and 144 g glycine in 400 mL of ultrapure water until fully dissolved.
• Up to 1 L with ultrapure water.
• Store at 4℃ and dilute to 1X when preparing for electrophoresis.
Tris-MES-SDS ( 20X ) 50 mM MES
50 mM Tris Base
0.1 % SDS
1 mM EDTA
pH 7.3		 • 97.6 g MES, 60.6 g Tris, 10 g SDS, and 3 g EDTA in 400 mL of ultrapure water.
• Up to 500 mL with ultrapure water.
• Store at 4℃ and dilute to 1X when preparing for electrophoresis.
Tris-MOPS-SDS ( 1X ) 50 mM MOPS
50 mM Tris Base
0.1% SDS
1 mM EDTA
pH 7.7		 • 104.6 g MOPS, 60.6 g Tris, 10 g SDS, and 3 g EDTA in 400 mL of ultrapure water.
• Up to 500 mL using ultrapure water.
• Store at 4℃ and dilute to 1X when preparing for electrophoresis.
Tris-Tricine-SDS ( 20X ) 50 mM Tricine
50 mM Tris Base
0.1 % SDS
pH 8.2		 • 89.5 g Tricine, 60.6 g Tris, 10 g SDS, and 3 g EDTA in 400 mL of ultrapure water.
• Up to 500 mL using ultrapure water.
• Store at 4℃ and dilute to 1X when preparing for electrophoresis.
Tips: When preparing a non-denaturing buffer, simply follow the above formulas without adding SDS.
4、Loading Buffer

Loading Buffer is indispensable in various experiments. Based on the type of experiment, different buffers can be chosen.

Below, we present the preparation methods for DNA Loading Buffer, RNA Loading Buffer, and SDS-PAGE Loading Buffer.

Type of Solution System Formula
DNA Loading Buffer ( 6X ) 30 mM EDTA
38 % ( V/V ) Glycerol
0.05 % ( W/V ) Xylene Cyanol FF
0.05 % ( W/V ) Bromophenol Blue		 • 4.4 g EDTA, 250 mg Bromophenol Blue, and 250 mg Xylene Cyanol FF in 500 mL beaker.
• Add 200 mL of ultrapure water to the beaker, then heat and stir until fully dissolved.
• Add 180 mL of Glycerol and adjust the pH to 7.0 using 2 N NaOH.
• Up to 500 mL with ultrapure water, store it at room temperature.
RNA Loading Buffer ( 10X ) 10 mM EDTA
50 % ( V/V ) Glycerol
0.25 %( W/V ) Xylene Cyanol FF
0.25 %( W/V ) Bromophenol Blue		 • 200 µL of 0.5 M EDTA (pH 8.0), 25 mg Bromophenol Blue, and 25 mg Xylene Cyanol FF in 10 mL centrifuge tube.
• Add 4 mL of DEPC water to the centrifuge tube and stir until fully dissolved.
• Add 5 mL of Glycerol and mix thoroughly.
• Up to 10 mL with DEPC water, store it at room temperature.
SDS-PAGE Loading Buffer ( 5X ) 250 mM Tris-HCl ( pH 6.8 )
10 % ( W/V ) SDS
0.5 % ( W/V ) BPB
50 % ( V/V ) Glycerol
5 % ( W/V ) β-mercaptoethanol		 • 1.25 mL of 1 M Tris-HCl (pH 6.8), 0.5 g SDS, 25 mg BPB, and 2.5 mL Glycerol in 10 mL centrifuge tube.
• Up to 5 mL using ultrapure water in the centrifuge tube.
• After subdividing into 500 µL aliquots, store at room temperature.
• Prior to use, add 25 µL of β-mercaptoethanol.
Note: Loading buffer with added β-mercaptoethanol can be stored at room temperature for approximately one month.
5、Commonly used solutions for molecular hybridization

Molecular hybridization involves both nucleic acid and protein hybridization. In the experimental process, various commonly used solutions are an indispensable part. When using the membrane hybridization method, you can opt for Denhardt's solution. For denaturation and washing in nucleic acid hybridization, choices could be SSC buffer or DNA denaturation buffer.

Type of Solution System Formula
SSC buffer ( 20X ) 3 M NaCl
0.3 M Sodium citrate		 • 175.3 g NaCl and 88.2 g Sodium citrate dihydrate in 800 mL of ultrapure water.
• Add drops of 14 N HCl to adjust the pH to 7.0, then up to 1 L with ultrapure water.
• Sterilize using high pressure and store at room temperature.
Denhardt's Solution ( 50X ) 1 % ( W/V ) Ficoll 400
1 % ( W/V ) Polyvinylpyrrolidone
1 % ( W/V ) BSA		 • 5 g Ficoll 400, 5 g Polyvinylpyrrolidone, and 5 g BSA in 500 mL beaker.
• Add 400 mL of ultrapure water and stir until fully dissolved.
• Up to 500 mL with ultrapure water.
• After filtration through a 0.45 μm filter, divide into 25 mL aliquots and store at -20°C.
DNA Denaturation Buffer 1.5 M NaCl
0.5 M NaOH		 • 87.7 g NaCl and 20 g NaOH in 1 L beaker.
• Add 800 mL of ultrapure water to the beaker and stir until fully dissolved.
• Up to 1 L with ultrapure water, store it at room temperature.
6、Commonly used solutions for protein cleaning and transfer membranes

Protein analysis procedures involve the use of both transfer and cleaning fluids. Specifically, WB transfer buffer is used to move the blot from gel to a membrane. TBST buffer has multiple applications. It can be utilized for preparing blocking solutions, as well as primary or secondary antibodies. Moreover, it is employed for washing after antibody incubation in various experiments, including WB, IF, and IHC. WB blocking buffer is employed to minimize non-specific antibody binding.

Type of Solution System Formula
SSC buffer ( 20X ) 39 mM Glycine
48 mM Tris
0.037 % ( W/V ) SDS
20 % (V/V) Methanol		 • 2.9 g Glycine, 5.8 g Tris, and 0.37 g SDS in a 1 L beaker.
• Add 600 mL of ultrapure water to the beaker and dissolve completely.
• Up to 800 mL with ultrapure water, add 200 mL of methanol, and store at room temperature.
TBST buffer 20 mM Tris-HCl
150 mM NaCl
0.05 % ( W/V ) Tween 20		 • 8.8 g NaCl and place 20 mL 1 M Tris-HCl (pH 8.0) in a 1 L beaker.
• Add 800 mL of ultrapure water to the beaker and dissolve completely.
• Add 0.5 mL Tween 20 and mix thoroughly.
• Up to 1 L with ultrapure water, and store at 4℃.
WB blocking buffer 5 % ( W/V ) skimmed milk powder / TBST buffer • Add 5 g skim milk powder to 100 mL of TBST buffer, stir thoroughly to dissolve.
• Store at 4℃, Prepare before use.
7、Commonly used balanced salt solutions for cell culture

Balanced salt solutions are common solutions used in cell culture processes. When choosing balanced salt solutions, PBS is mainly used for rinsing, freezing, and cultivating embryos, tissues, and cells. In addition to the uses of PBS, HBSS can also be used for the preparation of cell culture reagents and the dilution of cell counting. EBSS has the strongest buffering capacity and is designed specifically for the short-term maintenance of cells in a CO2 environment.

Type of Solution System Formula
PBS ( 10X ) 10.6 mM KH2PO4
1552 mM NaCl,
30 mM Na2HPO4·7H2O
pH 7.4		 • 1.44 g KH2PO4
• 90 g NaCl
• 8.0 g Na2HPO4·7H2O
( 1L )
HBSS (1X, Ca2+, Mg2+, phenol red ) 1.3 mM CaCl2
0.5 mM MgCl2·6H2O
0.4 mM MgSO4·7H2O
5.3 mM KCl,
0.4 mM, KH2PO4
4.1 mM NaHCO3
138 mM NaCl
0.3 mM Na2HPO4
5.5 mM D-Glucose
0.025 mM Phenol Red		 • 0.14 g CaCl2
• 0.1 g MgCl2·6H2O
• 0.1 g MgSO4·7H2O
• 0.4 g KCl
• 0.06 g KH2PO4
• 0.35 g NaHCO3
• 8 g NaCl
• 0.05 g Na2HPO4
• 1 g D-Glucose
• 0.01 g Phenol Red
(1L)
EBSS ( 1X，Ca2+, Mg2+, phenol red ) 1.8 mM CaCl2
0.8 mM MgSO4·7H2O
5.3 mM KCl
26 mM NaHCO3
117 mM NaCl
1 mM NaH2PO4·H2O
5.5 mM D-Glucose
0.025 mM Phenol Red		 • 0.2 g CaCl2
• 0.2 g MgSO4-7H2O
• 0.4 g KCl
• 2.2 g NaHCO3
• 6.8 g NaCl
• 0.14 g NaH2PO4·H2O
• 1 g D-Glucose
• 0.01 g Phenol Red
（1L）
8、Commonly used solutions for microbial culture

In microbial culture, LB medium is the most commonly used. Additionally, Ampicillin serves as a means to select resistant bacteria. IPTG is typically employed in blue-white screening. Meanwhile, X-Gal , a chromogenic substrate for β-galactosidase , is regularly utilized in situ staining of β-galactosidase and in blue-white screening.

Type of Solution System Formula
Ampicillin (100 mg/mL ) 100 mg/mL Ampbillin • 5 g Ampbillin into 50 mL centrifuge tube.
• Add 40 mL of ultrapure water to fully dissolve and up to 50 mL.
• Sterilize by filtration using a 0.22 μm filter membrane.
• Aliquot into small portions (1 mL) and store at -20℃.
IPTG (24 mg/mL ) 24 mg/mL IPTG • 1.2 g IPTG into 50 mL centrifuge tube.
• Add 40 mL of ultrapure water to fully dissolve and up to 50 mL.
• Sterilize by filtration using a 0.22 μm filter membrane.
• Aliquot into small portions (1 mL) and store at -20℃.
X-Gal(20 mg/mL ) 20 mg/mL X-Cal • 1 g of X-Cal into 50 mL centrifuge tube.
• Add 40 mL DMF to dissolve and up to 50 mL.
• Aliquot into small portions (1 mL) and store at -20℃.
LB medium 1 % ( W/V ) Tryptone
0.5 % ( W/V ) Yeast Extract
1 % ( W/V ) NaCl		 • 10 g Tryptone, 5 g Yeast Extract, and 10 g NaCl in a 1 L beaker.
• Add 800 mL of ultrapure water and stir thoroughly to dissolve.
• Add 5 N NaOH (approximately 0.2 mL) dropwise to adjust the pH to 7.0.
• Up to 1 L with ultrapure water.
• After high temperature and high pressure sterilization, store at 4℃.
Advanced Knowledge Points of Buffer Solutions

A Buffer Solution, typically a mixture composed of weak acids and their salts or weak bases and their salts, is an important concept in analytical chemistry. The pH of a buffer solution can remain stable within a certain range, unperturbed by dilution or the addition of small amounts of acid or base. This stability makes it widely applicable in various types of experiments.

How to choose a buffer?

Select a buffer based on the experiment's required pH range. A buffer, a mix of weak acids, bases and their salts, needs adequate buffering capacity. Ideally, the pKa of its weak acid should match or be near the experiment's required pH. pKa is crucial in buffer selection.

Table 1. Buffering range and pKa of common buffers
Buffer Name Molecular Weight Effective pH range pKa (20 °C) pKa (25 °C) pKa (37 °C)
MES 195.24 5.5–6.7 6.16 6.10 5.97
Bis-Tris 209.24 6.3-9.5 N/A 6.50 6.36
HEPES 238.30 6.8-8.2 7.55 7.48 7.31
MOPS 231.25 6.5-7.9 7.28 7.20 7.02
CHES 207.29 8.6-10.0 9.55 9.49 9.36
PIPES 302.37 6.1-7.5 7.28 7.17 7.14
ADA 190.15 6.0-7.2 7.01 6.88 6.84
BES 213.25 6.4-7.8 7.49 7.26 7.19
Tris 121.14 7.0-9.1 N/A 8.10 N/A
Tricine 179.17 7.4-8.8 8.54 8.23 8.14
FAQs about Buffer Preparation
Scenes Analyze
When the prepared buffer solution is different from the specified value, in order to make the buffer solution reach the specified pH value, readjust it with strong acid or strong base such as HCl or NaOH. Although the pH value of the buffer treated in this way is adjusted correctly, its buffer system is also destroyed.
Use the buffer after it has been stored for a long time or not properly stored. Many buffer solutions contain volatile components. Long-term storage can easily lead to solution failure. The buffer solution should be prepared in an appropriate amount and stored in a closed container at low temperature after use.
Summary

In this edition, MCE compiles common lab buffer preparation methods, saving you the time of repeated referencing during experiments. Come and collect the article.

Products Recommended

MES

MES is broadly used to regulate pH value for plants culture medium, reagent solution, and physiological experiments.

Tris

Tris is a biologically inert amino alcohol of low toxicity, which buffers carbon dioxide and acids in vitro and in vivo.

Bis-Tris

Suitable for hemoglobin separation.

HEPES

HEPES, a nonvolatile zwitterionic chemical buffering agent, is broadly applied in cell culture.

MOPS

MOPS sodium salt is commonly used as a buffering agent in biology.

Tricine

Tricine is widely used as a buffer in protein electrophoresis, nucleic acid analysis, and immunology, which can help adjust pH and stabilize chemical reactions.

EDTA

EDTA has antibacterial, anti-inflammatory, antioxidant, anti-hypercalcemia and anticoagulant activities.

ADA

ADA is a biological buffer. ADA can serve as a chelator.

PIPES

PIPES is an important component of PIPES buffer agent used in biochemistry.

BES

BES is used in the diagnostic testing area.

Boric acid

Boracic acid is an antiseptic agent. Used to prepare biological buffers.

Glycine

Glycine can be used to study cell protection, cancer, neurological diseases, and angiogenesis.

Tryptone

Tryptone is a common component of biological media.

Yeast extract

Yeast extract is a concentrate of the soluble part of yeast, especially Saccharomyces cerevisiae.

IPTG

Can be used for blue and white spot screening.

X-GAL

X-GAL is a widely used chromogenic β-galactosidase substrate.