Category Archives: Solution Preparation

Preparation of Neutralization solution (solution III) for the isolation of plasmid by alkaline lysis method

Overview

  • Neutralization solution (solution III) is used for the isolation of plasmid DNA by alkaline lysis method.
  • Neutralization solution is nothing but a potassium acetate solution which has pH 4.8.
  • Addition of neutralization solution in lysed bacterial cells brings the pH back, resulting in precipitation of protein and genomic DNA.
  • Both plasmid and genomic DNA renatures upon addition of neutralization buffer. While plasmid DNA renatures in correct conformation due to its circular and covalent nature, therefore, remains in the solution, genomic DNA precipitates due to random association of both the strands.
  • Sodium dodecyl sulfate (SDS) of the lysis buffer reacts with Potassium acetate and form insoluble Potassium dodecyl sulfate (KDS).

Requirements

  • Reagents
    • 5 M Potassium acetate (CH3CO2K) solution
    • Glacial acetic acid
    • Deionized / Milli-Q water
  • Equipment and disposables
    • Measuring cylinder
    • Conical flask / Beaker
    • Magnetic stirrer (optional)

Composition

  • 3 M Potassium
  • 5 M Acetate

Objective:

Preparation of 100 ml of Neutralization solution (solution III)

Preparation:

Step 1: To prepare, 100 ml of Neutralization solution, take 28.5 ml of Deionized / Milli-Q water in a 100 ml measuring cylinder.

Step 2: Add 60 ml of 5 M Potassium acetate and 11.5 ml of glacial acetic acid. Mix the solution.

Storage
  • Solution can be stored at room temperature in a tightly closed bottle for 1 year.
Applications
  • Plasmid isolation by alkaline lysis method
Follow the table To prepare Neutralization solution of various volume (10 ml, 25 ml, 50 ml and 1,00 ml).
Reagents / Volume 10 ml 25 ml 50 ml 100 ml
5 M Potassium acetate 6.0 ml 15 ml 30 ml 60 ml
Glacial acetate acid 1.15 ml 2.875 ml 5.75 ml 11.5 ml
Water 2.85 ml 7.13 ml 14.25 ml 28.5 ml

Preparation of Lysis solution (solution II) for the isolation of plasmid by alkaline lysis method

Overview

  • Lysis solution (solution II) is used for the isolation of plasmid DNA by alkaline lysis method.
  • The plasmid-containing bacterial cells are lysed by treatment with the lysis solution.
  • Lysis solution contains sodium hydroxide (NaOH) and sodium dodecyl sulfate (SDS).
  • SDS is a detergent which solubilizes the phospholipid and denatures the protein, leading to lysis and release of the cell contents. Denaturing action of SDS also releases protein from DNA, leaving the DNA (both genomic and plasmid DNA) free from proteins.
  • High alkaline condition due to NaOH denatures the plasmid and genomic DNA.

Requirements

  • Reagents and solutions

    • 10 N Sodium hydroxide (NaOH) solution
    • 10% sodium dodecyl sulfate (SDS)
    • Deionized / Milli-Q water
  • Equipment and disposables
    • Measuring cylinder
    • Conical flask / Beaker
    • Magnetic stirrer (optional)
Composition
  • 0.2 N Sodium hydroxide (NaOH)
  • 1% (wt/vol) Sodium Dodecyl Sulfate (SDS)

Objective

  • Preparation of 10 ml of lysis solution (solution II)

Preparation

Step 1: To prepare, 10 ml of lysis solution, take 8 ml of Deionized / Milli-Q water in a 10 ml measuring cylinder.

Step 2: Add 0.2 ml of 10 N NaOH solution and 1.0 ml of 10% sodium dodecyl sulfate (SDS).

Tip:
  • You may see some white precipitate when you add SDS. Dissolve it by mixing.
Precaution:
  • Do not mix concentrated stock solutions together. This will cause precipitation.

Step 3: Adjust the volume to 10 ml with deionized / Milli-Q water. Mix the solution.

Storage
  • Solution can be stored at room temperature for a week. It is recommended to prepare fresh lysis solution for optimal lysis.
Applications
  • Preparation of plasmid DNA by alkaline lysis method
Follow the table to prepare lysis solution of various volume (10 ml, 25 ml, 50 ml and 1,00 ml).
Reagents / Volume 10 ml 25 ml 50 ml 100 ml
10 N Sodium hydroxide (NaOH) 0.2 ml 0.5 ml 1 ml 2 ml
10% sodium dodecyl sulfate (SDS) 1.00 ml 2.5 ml 5 ml 10 ml
Water 8.8 ml 22 ml 44 ml 88 ml

Preparation of resuspension buffer for the isolation of plasmid by alkaline lysis method

Overview:

  • Resuspension buffer is used to resuspend bacterial cells during plasmid isolation by alkaline lysis method. It provides an optimal starting pH (pH 8.0) and an ideal condition for subsequent lysis.
  • Resuspension buffer containing Tris and EDTA is very common.
  • Tris.Cl acts as a buffering agent and maintains the pH of the resuspension buffer 8.0.
  • EDTA  chelates the divalent cations which are released upon bacterial lysis. Divalent cations are required for many enzymatic reactions. EDTA action results in inactivation of many enzymes which may harm plasmid DNA.
  • Resuspension buffer can be supplemented with RNase A which helps to get rid of RNA contamination from the plasmid preparation.

To know more, please read the article: Resuspension buffer (solution I) for isolation of plasmid by alkaline lysis method.

Requirements

  • Reagents
    • 1M Tris.Cl (pH 8.0) solution, autoclaved
    • 0.5 EDTA (pH 8.0) solution, autoclaved
    • Deionized / Milli-Q water
  • Equipment and disposables
    • Measuring cylinder
    • Conical flask / Beaker
    • Magnetic stirrer (optional)

Composition

  • 25 mM Tris.Cl (pH 8.0)
  • 10 mM EDTA (pH 8.0)

Objective

Preparation of 100 ml of resuspension buffer (solution I)

Preparation

Step 1: To prepare 100 ml of resuspension buffer, take 95.5 ml of deionized / Milli-Q water in a 100 ml measuring cylinder/beaker.
Precaution:
  • Do not mix concentrated stock solutions together. This can cause precipitation.
Step 2: Add 2.5 ml of Tris.Cl (pH 8.0) and 2.0 ml of EDTA (pH 8.0). Mix and transfer to a transparent bottle.
Tip:
  • A transparent bottle can easily be examined for any microbial growth in resuspension buffer.

Storage

The solution can be stored at 4°C for 6 months.

Precaution:
  • Frequently check the presence of any microbial growth in resuspension buffer. Discard if you detect any microbial growth.

Application

Preparation of plasmid DNA by alkaline lysis method

Follow the table to prepare resuspension buffer of various volume.
Reagents / Volume 10 ml 25 ml 50 ml 100 ml
1M Tris.Cl (pH 8.0) 0.25 ml 0.625 ml 1.25 ml 2.5 ml
0.5 M EDTA (pH 8.0) 0.2 ml 0.5 ml 1.0 ml 2.0 ml
Water 9.45 ml 23.625 ml 47.25 ml 95.5 ml

 

Preparation of RNase A containing resuspension buffer for the isolation of plasmid by alkaline lysis method

Overview:

  • Resuspension buffer is used to resuspend bacterial cells during plasmid isolation by alkaline lysis method. It provides an optimal starting pH (pH 8.0) and an ideal condition for subsequent lysis.
  • Resuspension buffer can be supplemented with RNase A. RNase A is a very stable enzyme and is active under the very stringent condition including high alkaline condition, the presence of detergents and chelating agents (EDTA, CDTA).
  • RNase A digest RNAs which are released from bacteria during lysis step, thus allow plasmid preparation free from RNA contamination. However, such plasmid preparation cannot be used for in-vitro transcription due to contamination of RNases.

To know more, please read the article: Resuspension buffer (solution I) for isolation of plasmid by alkaline lysis method.

Requirements

  • Reagents
    • 1M Tris.Cl (pH 8.0) solution, autoclaved
    • 0.5 EDTA (pH 8.0) solution, autoclaved
    • 10 mg/ml RNase A
    • Deionized / Milli-Q water
  • Equipment and disposables
    • Measuring cylinder
    • Conical flask / Beaker
    • Magnetic stirrer (optional)

Composition

  • 25 mM Tris.Cl (pH 8.0)
  • 10 mM EDTA (pH 8.0)
  • 100 μg/ml RNase A

Objective

Preparation of 100 ml of resuspension buffer (solution I)

Preparation

Step 1: To prepare 100 ml of resuspension buffer, take 94.5 ml of deionized / Milli-Q water in a 100 ml measuring cylinder/beaker.
Precaution:
  • Do not mix concentrated stock solutions together. This can cause precipitation.
Step 2: Add 2.5 ml of Tris.Cl (pH 8.0), 2.0 ml of EDTA (pH 8.0), and 1 ml RNase A. Mix and transfer to a transparent bottle.
Tip:
  • A transparent bottle can easily be examined for any microbial growth in resuspension buffer.

Storage

The solution can be stored at 4°C for 3 – 6 months.

Precaution:
  • Frequently check the presence of any microbial growth in resuspension buffer. Discard if you detect any microbial growth.

Application

Preparation of plasmid DNA by alkaline lysis method

Follow the table to prepare resuspension buffer of various volume.
Reagents / Volume 10 ml 25 ml 50 ml 100 ml
1M Tris.Cl (pH 8.0) 0.25 ml 0.625 ml 1.25 ml 2.5 ml
0.5 M EDTA (pH 8.0) 0.2 ml 0.5 ml 1.0 ml 2.0 ml
10 mg/ml RNase A 0.1 ml 0.25 ml 0.5 ml 1.0 ml
Water 9.45 ml 23.625 ml 47.25 ml 94.5 ml

Preparation of glucose and RNase A containing resuspension buffer for the isolation of plasmid by alkaline lysis method

Overview:

  • Glucose-containing resuspension buffer is used to resuspend bacterial cells during plasmid isolation by alkaline lysis method.
  • Glucose is added to make the resuspension buffer isotonic. However, isotonicity is not required for cell wall containing bacteria including E. coli DH5α. Cell wall containing bacteria can withstand a wide range of solution concentration.
  • Glucose-containing resuspension buffers are prone to microbial growth, therefore cannot be stored for a long time, and need to be kept at 4°C.
  • Resuspension buffer can be supplemented with RNase A. RNase A is a very stable enzyme and is active under the very stringent condition including high alkaline condition, the presence of detergents and chelating agents (EDTA, CDTA).
  • RNase A digest RNAs which are released from bacteria during lysis step, thus allow plasmid preparation free from RNA contamination. However, such plasmid preparation cannot be used for in-vitro transcription due to contamination of RNases.

To know more, please the read article: Preparation of resuspension buffer (solution I) for isolation of plasmid by alkaline lysis method.

Requirements

  • Reagents
    • 1M Glucose solution, filter sterilized
    • 1M Tris.Cl (pH 8.0) solution, autoclaved
    • 0.5 EDTA (pH 8.0) solution, autoclaved
    • 10 mg/ml RNase A
    • Deionized / Milli-Q water
  • Equipment and disposables
    • Measuring cylinder
    • Conical flask / Beaker
    • Magnetic stirrer (optional)

Composition

  • 50 mM Glucose
  • 25 mM Tris.Cl (pH 8.0)
  • 10 mM EDTA (pH 8.0)
  • 100 μg/ml RNase A

Objective

Preparation of 100 ml of resuspension buffer (solution I)

Preparation

Step 1: To prepare 100 ml of resuspension buffer, take 89.5 ml of deionized / Milli-Q water in a 100 ml measuring cylinder/beaker.
Precaution:
  • Do not mix concentrated stock solutions together. This can cause precipitation.
Step 2: Add 5 ml of 1 M glucose solution, 2.5 ml of Tris.Cl (pH 8.0), 2.0 ml of EDTA (pH 8.0), and 1 ml RNase A. Mix and transfer to a transparent bottle.
Tip:
  • A transparent bottle can easily be examined for any microbial growth in resuspension buffer.

Storage

The solution can be stored at 4°C for 3 – 6 months.

Precaution:
  • Frequently check the presence of any microbial growth in resuspension buffer. Discard if you detect any microbial growth.

Application

Preparation of plasmid DNA by alkaline lysis method

Follow the table to prepare resuspension buffer of various volume.
Reagents / Volume 10 ml 25 ml 50 ml 100 ml
1M Glucose solution 0.5 ml 1.25 ml 2.5 ml 5 ml
1M Tris.Cl (pH 8.0) 0.25 ml 0.625 ml 1.25 ml 2.5 ml
0.5 M EDTA (pH 8.0) 0.2 ml 0.5 ml 1.0 ml 2.0 ml
10 mg/ml RNase A 0.1 ml 0.25 ml 0.5 ml 1.0 ml
Water 8.95 ml 22.375 ml 44.75 ml 89.5 ml

Preparation of glucose containing resuspension buffer for the isolation of plasmid by alkaline lysis method

Overview:

  • Glucose-containing resuspension buffer is used to resuspend bacterial cells during plasmid isolation by alkaline lysis method.
  • Glucose is added to make the resuspension buffer isotonic. However, isotonicity is not required for cell wall containing bacteria including E. coli DH5α. Cell wall containing bacteria can withstand a wide range of solution concentration.
  • Glucose-containing resuspension buffers are prone to microbial growth, therefore cannot be stored for a long time, and need to be kept at 4°C.

To know more, please read the article: Resuspension buffer (solution I) for isolation of plasmid by alkaline lysis method.

Requirements

  • Reagents
    • 1M Glucose solution, filter sterilized
    • 1M Tris.Cl (pH 8.0) solution, autoclaved
    • 0.5 EDTA (pH 8.0) solution, autoclaved
    • Deionized / Milli-Q water
  • Equipment and disposables
    • Measuring cylinder
    • Conical flask / Beaker
    • Magnetic stirrer (optional)

Composition

  • 50 mM Glucose
  • 25 mM Tris.Cl (pH 8.0)
  • 10 mM EDTA (pH 8.0)

Objective:

Preparation of 100 ml of resuspension buffer (solution I) containing glucose

Preparation:

Step 1: To prepare 100 ml of resuspension buffer, take 90.5 ml of deionized / Milli-Q water in a 100 ml measuring cylinder/beaker.
Precaution:
  • Do not mix concentrated stock solutions together. This can cause precipitation.
Step 2: Add 5 ml of 1 M glucose solution, 2.5 ml of Tris.Cl (pH 8.0) and 2.0 ml of EDTA (pH 8.0). Mix and transfer to a transparent bottle.
Tip:
  • A transparent bottle can easily be examined for any microbial growth in resuspension buffer.

Storage

The solution can be stored at 4°C for 6 months.

Precaution:
  • Frequently check the presence of any microbial growth in resuspension buffer. Discard if you detect any microbial growth.

Application:

Plasmid isolation from E.coli by alkaline lysis method

Follow the table to prepare resuspension buffer of various volume.
Reagents / Volume 10 ml 25 ml 50 ml 100 ml
1M Glucose solution 0.5 ml 1.25 ml 2.5 ml 5 ml
1M Tris.Cl (pH 8.0) 0.25 ml 0.625 ml 1.25 ml 2.5 ml
0.5 M EDTA (pH 8.0) 0.2 ml 0.5 ml 1.0 ml 2.0 ml
Water 9.05 ml 22.625 ml 45.25 ml 90.5 ml

Resuspension buffer (solution I) for isolation of plasmid by alkaline lysis method

  • Resuspension buffer (solution I) is used for the isolation of plasmid DNA by alkaline lysis method. Bacterial cells, obtained from the culture (liquid culture or colonies, grown on a agar plate), is resuspended in this buffer. The purpose of resuspension buffer is to provide an optimal starting pH (pH 8.0) and an ideal condition for subsequent lysis.
  • The classical composition of resuspension buffer (designed by Birnboim and Doly) contained Lysozyme, Glucose, Tris.Cl, and CDTA (or EDTA). Most of the recent formulations do not contain lysozyme and glucose.
  • Lysozymes are glycoside hydrolases that destroy bacterial cell walls by catalyzing the hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan. However, for most bacteria including E. coli DH5α, lysis solution was found to induce complete lysis, thus eliminated the use of lysozymes.
  • Glucose is added to make the solution isotonic. However, isotonicity is not required for cell wall containing bacteria including E. coli DH5α. Cell wall containing bacteria can withstand a wide range of solution concentration. Therefore, Glucose is not included in the recent recipes of resuspension buffer. Furthermore, glucose-containing resuspension buffer cannot be stored for a long time, and need to be kept at 4°C.
  • EDTA (or CDTA) chelates the divalent cations which are released upon bacterial lysis. Divalent cations are required for many enzymatic reactions. EDTA action results in inactivation of many enzymes which may harm plasmid DNA.
  • Tris.Cl acts as a buffering agent and maintains the pH of the resuspension buffer 8.0.
  • Now researchers prefer to supplement resuspension buffer with RNase A. RNase A is a very stable enzyme and is active under the very stringent condition including high alkaline condition, the presence of detergent and chelating agent (EDTA). Addition of RNase A in resuspension buffer helps to remove RNA from the plasmid preparation. In the subsequent lysis step, RNase A digests the RNA of the bacteria. However, such plasmid preparation cannot be used for in-vitro transcription due to contamination of RNases. In addition, RNase A containing resuspension buffer should be stored at 4°C and has a limited life (3 – 6 months).
  • pH indicator, LyseBlue from Qiagen, can also be added to the resuspension buffer. LyseBlue ensures the complete lysis and subsequently neutralization step. Both steps are very important to get high quality of plasmid DNA.
  • Resuspension buffer is not included in the protocol of plasmid isolation using plasmid isolation kit provided by some manufacturers (see Zyppy Plasmid Miniprep Kit). In those procedures, highly concentrated lysis buffer is added directly to the overnight grown liquid culture of bacterial cells.
  • The following types of resuspension buffer can be used for plasmid isolation

Preparation of freezing medium containing DMSO and FBS

Overview:

  • Freezing medium can be used to preserve cell lines for long term at ultra-low temperature. This method of preserving cell line is called cryopreservation.
  • An ideal freezing medium must allow 100% recovery without causing any change in cell characteristics after revival.
  • Freezing medium is nothing but a complete medium supplemented with high concentration of serum and a cryoprotective agent such as DMSO or glycerol.
  • As the name suggest, cryoprotective agents protect cells from lysis due to ice crystal formation at temperature below freezing point.
  • Serum concentration as high as 90% can be used in freezing medium. High serum concentration improves cell viability and recovery after thawing.
  • Serum-free chemically-defined freezing media are also available. They are very useful to preserve those cell lines which are maintained in serum-free chemically defined medium.
  • Both serum-containing and serum-free freezing medium are available commercially.
  • Serum-containing medium is used for cell lines which are maintained in serum-supplemented growth medium. We recommend to check cell line manual for optimal freezing medium.

Requirements

  • Reagents
    • Fetal bovine serum (FBS) or Fetal calf serum (FCS)
    • DMSO (sterile)
    • Complete growth medium (optional)
  • Equipment and disposables
    • 50 ml sterile polypropylene tubes
    • Pipetboy
    • Pipets
    • Laminar flow hood
Note:
  • Use the same growth medium which is used to maintain cell line (e.g., use DMEM, if the cell line is maintained in DMEM medium).

Composition:

  • 50% FBS
  • 40% complete growth medium
  • 10% DMSO

Objective:

Preparation of 50 ml serum-containing freezing medium

Note: All operations must be done under sterile condition. Wipe carefully surface of reagent containing bottles (e.g., DMSO bottle, FBS bottle) with 70% ethanol before placing them inside flow hood.

Preparation:

Step 1: To prepare 50 ml freezing medium, transfer 25 ml FBS and 20 ml growth medium to a 50-ml sterile polypropylene tube. Tighten the cap of the tube and mix by gentle inversion. Store it on ice.

Tips: You can also use sterile containers like beaker. We recommend you to use 50 ml sterile polypropylene tube.

Step 2: Step 2: Add 5 ml DMSO slowly while shaking the tube. Tighten the cap of the tube and mix by inverting the tube many times (5 – 6 times).
Note: Mixing of DMSO to serum is an exothermic reaction which can cause denaturation of serum proteins, resulting in precipitation. Therefore, it is recommended to use chilled FBS.

Precautions: Do not store DMSO on ice. It will solidify on ice.

Step 3 (optional): Check the sterility of freezing by keeping a small aliquot in a petri dish in the CO2 incubator.
Tips: We recommend you to check sterility of freezing medium.

Storage:

Store at 4°C for few days. Freezing medium is stable for at least 6 months at -20°C.

Tips: Freezing and thawing multiple times can cause proteins denaturation and precipitation.

Applications:

  • Serum-containing freezing medium is used for cryopreservation of cell lines.

Follow the table to prepare freezing medium of different volume.

Reagent/volume 10 ml 50 ml 100 ml 250 ml
FBS 5 25 50 125
DMSO 1 5 10 25
Growth medium 4 20 40 100

Preparation of 6X DNA loading dye (Bromophenol blue, xylene cyanol FF and Ficoll)

 Overview

  • DNA sample is mixed with DNA loading dye prior to loading into the wells of agarose gel.
  • Two tracking dyes containing DNA loading dye is very common for DNA gel electrophoresis. The most common tracking dyes are bromophenol blue and xylene cyanol FF.
  • Bromophenol blue (C19H10Br4O5S ; Molar mass – 669.96 gram/mole) is a weak acid. It is available commercially as a light pink to purple crystalline powder.
  • The color of aqueous solution of bromophenol blue is pH dependent. Bromophenol blue solution appears yellow at pH 3.0, purple at pH 4.6, and blue at neutral pH.
  • Xylene cyanol FF (C25H27N2NaO6S2 ; Molar mass – 538.61 gram/mole) is available commercially as a dark green crystalline powder.
  • Both tracking dyes, Bromophenol blue and Xylene cyanol FF, are soluble in water (solubility in water is ~ 1 mg/ml) and carry net negative charge at neutral or slightly basic pH (the pH of the electrophoresis buffer). The net negative charge on Xylene cyanol FF is less than Bromophenol blue. Because of this, Bromophenol blue move faster than xylene cyanol in agarose gel in spite of its higher molecular weight.
  • The composition of agarose gel affects moving position of bromophenol blue and xylene cyanol FF in the gel. The bromophenol blue and xylene cyanol FF co-migrates with ~300 bp and ~4000 bp DNA fragments in 1% agarose gel respectively.
  • Ficoll 400 is a high molecular weight, neutral, hydrophilic, polysaccharide. It is added to provide high density to sample.
  • 6X DNA loading dye containing bromophenol blue, Xylene cyanol FF and Ficoll 400 appears dark blue/ purple in color.
  • A 6X DNA loading dye can have tracking dye concentration ranging from 0.03% to 0.50% (W/V). High concentration provides very good contrast colour, which is easy to monitor upon electrophoresis progression. However, high tracking dye concentration masks the co-migrating DNA fragments, and interfere in the analysis of co-migrating DNA bands (e.g., densitometric analysis). Low concentration of tracking dye is preferred when DNA sample is expected to contain co-migrating DNA fragment(s). However, low concentration of tracking dye causes a compromise in the visibility of migrating dye band, which sometime disappear after a long electrophoresis run.

Requirement

  • Reagents
    • Bromophenol blue
    • Xylene cyanol FF
    • Ficoll 400
    • Deionized / Milli-Q water
  • Equipment and disposables
    • Measuring cylinder
    • Conical flask / Beaker / 15 ml screw-capped tube
    • Magnetic stirrer (optional)

Composition of 6X DNA loading dye

  • 0.25% (W/V) bromophenol blue
  • 0.25% (W/V) xylene cyanol FF
  • 15% (W/V) Ficoll 400

Composition of 1X DNA loading dye

  • 0.042% (W/V) bromophenol blue
  • 0.042% (W/V) xylene cyanol FF
  • 2.5% (W/V) Ficoll 400

Objective

  • Preparation of 10 ml of 6X DNA loading dye containing bromophenol blue, xylene cyanol FF and Ficoll 400

Preparation

Step 1: To prepare 10 ml of 6X DNA loading dye, weigh out 25 mg bromophenol blue, 25 mg xylene cyanol FF and 1.5 gram Ficoll 400. Transfer them to a screw-capped tube (15 ml with millilitre marks). Add 7 ml deionized / Milli-Q water. Mix until all ingredients are dissolved completely.

Tips:

  • We recommend to use disposable DNases-free 15 ml screw-capped tube with millilitre marks. Transfer all the contents and mix by inverting the tube number of times or use rotator. Since the tube is marked, you don’t need to transfer the content to measuring cylinder. Transferring solution may not be convenient as the solution contains dye.

Precautions:

  • Use nuclease-free, autoclaved deionized / Milli-Q water and glasswares.

Step 2: Adjust the volume to 10 ml with deionized / Milli-Q water. Mix it again.

Tips:

  • We recommend you to give a short spin to 15 ml tube just before adjusting the volume 10 ml. Short spin will help to collect all solution which are adhered to sides and lid of the tube.
  • The solution will appear dark blue/purple in colour with no undissolved particles. If there is any undissolved particles in the solution, remove them by centrifuging the tube at 4000 – 5000 rpm for 10 min at room temperature.

 

Storage:

Store the solution at room temperature or 4°C for long time.

Tips:

  • Aliquot 1 ml in 1.5 ml eppendorf microcentrifuge tubes.

Applications

  • This solution is used for loading DNA samples onto nondenaturing gels.
To prepare 6X agarose gel loading dye of various volume (5 ml, 10 ml, 25 ml, 100), follow the table.
Reagents / Volume 5 ml 10 ml 25 ml 50 ml 100 ml
Bromophenol blue 12.5 mg 25 mg 62.5 mg 125 mg 250 mg
Xylene cyanol FF 12.5 mg 25 mg 62.5 mg 125 mg 250 mg
Ficoll 400 0.75 gm 1.5 gm 3.75 gm 7.5 gm 15 gm
Water Adjust the final volume to 5 ml Adjust the final volume to 10 ml Adjust the final volume to 25 ml Adjust the final volume to 50 ml Adjust the final volume to 100 ml