Monthly Archives: March 2017

Preparation of 50X TAE electrophoresis buffer

Overview

  • TAE is one of the very common electrophoresis buffers, used for agarose gel analysis of DNA.
  • It contains Tris, acetic acid and EDTA.
  • Tris-acetate provides electrical conductivity and maintains pH.
  • EDTA inhibits metal dependent nucleases by chelating the divalent cations (Ca2+, Mg2+), thus protecting the DNA from nucleases during the run.
  • TAE buffer has lower buffering capacity than TBE, therefore it’s use should be avoided for extended and repeated electrophoresis.
  • TAE buffer is suitable for applications where gel eluted DNA fragments need to be modified using DNA modifying enzymes. In such cases, use of TBE buffer should be avoided as the borate of TBE buffer inhibits many enzymes (e.g., DNA ligases).

Requirement

  • Reagents
    • Tris base (C4H11NO3, Molecular Weight: 121.14)
    • Glacial acetic acid (CH3COOH, Molecular Weight: 60.05)
    • 0.5 M EDTA stock solution (pH 8.0)
    • Deionized / Milli-Q water
  • Equipment and disposable
    • Measuring cylinder
    • Conical flask / Beaker
    • Magnetic stirrer

Composition of 50X TAE buffer (Stock Solution)

  • 2.0 M Tris acetate
  • 0.05 M EDTA
  • pH 8.2 – 8.4 (at 25°C)

Composition of 1X TAE buffer (Working Solution)

  • 40 mM Tris acetate
  • 1 mM EDTA
  • pH 8.2 – 8.4 (at 25°C)

Objective

  • Preparation of 1000 ml of 50X TAE electrophoresis buffer.

Preparation

Step 1: Weigh out 242 grams of Tris base and transfer it to 2 L beaker / conical flask. Add 750 ml deionized / Milli-Q water and mix until all Tris base dissolves completely.

Tips:

  • One can use manual shaking using a glass pipette to mix the ingredients. Magnetic stirrer makes the dissolving process automated and convenient.

Step 2: Add 100 ml of 0.5 M EDTA solution and 57.1 ml glacial acetic acid. Mix the solution again. Adjust pH to 8.3 if required.

Precautions:

Since pH is dependent on temperature, we recommend to adjust the solution pH at room temperature (25°C).

Step 3: Adjust the solution volume to 1000 ml with deionized / Milli-Q water. Mix the solution again.

Optional : One can filter the solution to remove any undissolved materials.

Step 4: Sterilize the solution by autoclaving (20 minutes at 15 lb/sq.in. (psi) from 121-124°C on liquid cycle).

Notes:

  • One can sterilize the solution by passing through 0.22 μ filter unit. Filter sterilization removes all suspended particles with size more than 0.22 μ which includes most bacteria their spores but not mycoplasma. Moreover, it does not inactivate enzyme activities (e.g., DNases). Autoclaving inactivates most enzymes except some (e.g., RNases) and kills most microorganisms including mycoplasma.

Tips:

  • Transfer the solution to autoclavable bottle before autoclaving.
  • Depending on the consumption, one can make small aliquots of solution.

Storage

  • Solution can be stored at 15 – 25 °C (room temperature) for several months.

Precautions:

  • Discard solution if there is a considerable amount of precipitates.

Applications

  • Agarose gel electrophoresis of DNA
To prepare 50X TAE electrophoresis buffer of various volume (100 ml, 250 ml, 500 ml and 1000 ml), follow the table.
Reagents / Volume 100 ml 250 ml 500 ml 1000 ml
Tris base 24.2 gm 60.5 gm 121 gm 242 gm
Glacial acetic Acid 5.71 ml 14.27 ml 28.55 ml 57.1 ml
0.5 M EDTA (pH 8.0) 10 ml 25 ml 50 ml 100 ml
Water Adjust to the final volume 100 ml Adjust to the final volume 250 ml Adjust to the final volume 500 ml Adjust to the final volume 1000 ml

 

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

 

DNA loading dye

  • DNA samples are mixed with DNA loading dye / buffer prior to loading into the wells of agarose gel for electrophoresis.
  • Usually a DNA loading dye contains at least one dye (orange G, bromophenol blue, xylene cyanol FF or bromocresol green) and a high density reagent (glycerol, sucrose or Ficoll 400).
  • EDTA and/or SDS can also added in the loading dye.
  • Ingredients are dissolved in water, but tris buffer can also be used in place of water. Tris containing DNA loading dye is called DNA loading buffer.
  • DNA loading dye/buffer serves the following purposes_ _ _ _
    • It provides high density to DNA sample. Due to high density, DNA sample settled at the bottom of the agarose wells. Nicely settled DNA form sharp and crisp band upon electrophoresis.
    • DNA loading dye/buffer imparts colour to DNA sample which allows easy monitoring of sample loading process. Sample overflow, cross-contamination, or leakage of wells can easily be monitored due to colour which otherwise would be very difficult with colourless sample.
    • Since dye color can be seen by naked eye, electrophoresis progression can be easily monitored by observing the migration of dyes which otherwise would be difficult because DNA migration can not be monitored by naked eye and DNA visualization requires staining and special instrument (ethidium bromide staining and uv torch).
  • There are several dyes (Bromophenol blue, Bromocresol green, Orange G, Xylene cyanol FF) which can be used for the preparation of DNA loading dye/buffer. An ideal dye, used for preparation of DNA loading dye/buffer, should have following characteristics.
    • It should not interact with the DNA or component of electrophoresis buffer and gel.
    • Like DNA, It should have negative charge and move towards the positive pole.
    • It should imparts contrast colour to DNA sample and its migration should easily be monitored visually.
    • It should not interfere with the analysis of DNA.
    • The size of the dye must be within the upper and lower resolution limit of the agarose gel (50 bp to 20 KB)
  • Two dye containing loading buffer (mostly bromophenol blue and xylene cyanol FF) is very common for DNA gel electrophoresis. In such loading buffer, One dye migrates fast and correspond to the migration of DNA fragment 50 – 500 bp, whereas other dye migrate comparatively slow and correspond to the migration of DNA fragment 4000 – 8000 bp.
  • Use of a specific dye mostly depends on the application and the size of the DNA fragment(s) to be analyzed by the gel electrophoresis. DNA band can be masked by co-migrating dye, which can give wrong information about the amount of DNA fragment. Depending on the size of DNA fragment, one should choose the dye.
  • The high density reagent (glycerol, sucrose or Ficoll 400) is added to facilitate the sample to be placed in the well of the gel. Due to high density, DNA sample settled at the bottom of the well. It also helps DNA sample to be confined in the well without diffusing out.
  • Since glycerol interacts with the borate of TBE electrophoresis buffer, glycerol containing loading dye / buffer are not recommended to use with TBE buffer.
  • Sucrose containing loading dye / buffer are prone to develop mold like growth. Therefore, sucrose containing loading dye / buffer can not be kept for long time at 4°C.
  • Ficoll-400 containing loading dyes / buffers can be stored at room temperature and also function as better density agent.
  • EDTA is added to inhibit the action of nucleases or DNA modifying enzymes which require divalent cation for their action.
  • Tris functions as a buffering agent and maintain the pH of the loading dye.
  • SDS reduces the DNA-protein interaction, thus helpful to run DNA samples containing proteins or enzymes (e.g., alkaline phosphatase).
  • DNA loading buffer is generally made in 6X concentration and mostly contains two dyes, bromophenol blue and xylene cyanol. Some DNA loading dyes contain only one dye while others may contain three dyes.

Plasmid isolation by alkaline lysis method (miniprep)

Overview

  • Alkaline lysis method of plasmid isolation was originally developed by Brinboim and Doly (1979).
  • In this procedure, bacteria containing the desired plasmid are harvested from liquid bacterial culture by centrifugation.
  • Suspension of bacteria is made in isotonic solution which is subsequently subjected to lysis by an alkaline solution containing a detergent, Sodium dodecyl sulfate (SDS), and an alkali, Sodium hydroxide (NaOH).
  • While sodium dodecyl sulfate serves to lyse cells and denature proteins, alkaline condition denatures genomic DNA, plasmid DNA and proteins.
  • Lysed cell mixture is further neutralized by Potassium acetate (pH 5.2). This results in renaturation of plasmid and genomic DNA. Since plasmid DNA is covalently closed, it reanneals while genomic DNA form precipitate.
  • Precipitate is separated by high speed centrifugation. Plasmid from the supernatant is recovered by precipitation using isopropanol or ethanol.

Requirement

  • Reagents
    • Resuspension Buffer (50 mM glucose, 25 mM TrisCl (pH 8.0), 10 mM EDTA, pH 8.0)
    • Lysis solution (0.2 N NaOH, 1% (wt/vol) SDS)
    • Neutralization solution (3 M potassium acetate, pH 4.8)
    • Phenol : Chloroform : Isoamyl alcohol (25 : 24 : 1) solution (Optional)
    • 70% Ethanol
    • Isopropanol
    • Tris – EDTA (TE) (100 mM Tris, 10 mM EDTA, pH 8.0)
    • DNase free RNase (10 mg/ml)
  • Equipment and disposables
    • Microcentrifuge tube
    • Micropipette and tips
    • Ice
    • Gloves

Objective: Isolation of plasmid DNA from 1-3 ml of bacterial culture (E. coli DH5α) by Alkaline lysis method.

Starting material: 3 ml overnight grown culture of E. coli DH5α containing plasmid of interest. (see protocol)

Prior to start:
  • Make sure that neutralization solution is chilled
  • Set the centrifuge for cooling (4°C)
  • Prepare the lysis solution freshly
  • Add DNase free RNase in resuspension buffer

Protocol

Step 1: Pour 1.5 ml overnight grown culture in a microcentrifuge tube. Centrifuge at room temperature (or 4°C) for 60 seconds at 12,000 rpm (or 5,000 rpm for 5 min). Remove the supernatant from the tube completely, leaving the bacterial pellet as dry as possible.
Notes:
  • The yield of plasmid DNA is dependent mainly on the copy number of plasmid. For high copy number plasmid, 1.5 ml culture is sufficient to get good yield of plasmid DNA. However, more culture is required for good yield of low-copy number plasmid.
Tips:
  • To remove the medium completely, decant the medium from the microcentrifuge tube after centrifugation. Invert microcentrifuge tube upside down on a paper towel to remove residual liquid. Gently tap the tube on the paper towel to remove liquid sticking on the sides of the tube.
  • To take more bacterial culture (more than 1.5 ml) for plasmid isolation, repeat the above process by adding more culture in the same microcentrifuge tube. Microcentrifuge tube with 2 ml capacity can also be used.
  • To increase the yield of low copy number plasmid DNA, one can collect the bacterial cells from 5 ml culture by repeating the above process thrice. Lysis may not be efficient in such cases due to high number of bacterial cells. In such situation, double the amount of all reagents (resuspension Buffer, lysis solution, and neutralization solution).
  • Chloramphenicol treatment can be used to amplify low-copy number plasmid.
  • The procedure may be stopped at this point and continued later by freezing the bacterial cell pellets. The bacterial cell pellets can be stored at -20°C for months or at -70°C for years.
Precautions:
  • While harvesting the bacteria, speed of centrifugation and time should be optimized in such a way that the pellet after centrifugation should be loose and at the same time supernatant should be clear. If pellet is tight, it would be difficult to make the suspension of the pellet. Generally above mentioned condition works well.
  • Try to remove medium from the pellet completely. Traces of medium may inhibit some of the sensitive restriction enzymes action. Additionally pellet can be washed with 200 μl of resuspension buffer. Generally washing is not required for miniprep.

Step 2: Add 100 μl ice cold resuspension buffer. Resuspend the bacterial pellet properly by vortexing or by slow rounds of pipetting with a 100 μl micropipette. Incubate on ice for 5 min.

Tips:
  • The procedure can be stopped at this point and continued later by freezing the suspension of bacterial cells. It can be stored at -20 °C for months or at -70 °C for years. We recommend to stop the procedure (if necessary) at this stage instead of previous step as making resuspension of frozen bacterial pellet is not so efficient. If you resuspend the bacterial pellet before freezing, you need to just thaw the content and proceed to next step.
  • To get rid of bacterial RNA, one can supplement resuspension buffer with RNase A (final conc. 100 μg/ml). RNases are very stable and retain their activity for long time under harsh conditions, like high alkaline denaturing condition. RNA are degraded upon lysis of cells by RNase A.

Precautions: Ensure that the bacterial pellet is completely dispersed in resuspension buffer prior to lysis. No cell clumps should be visible before the addition of Lysis Solution. Inefficient lysis results in low yield of plasmid.

Step 3: Add 200 μl of freshly prepared Lysis Solution to bacterial suspension. Close the tube tightly, and mix contents thoroughly by inverting and rolling the sealed tube 4 – 6 times until the solution becomes viscous and slightly clear. Incubate on ice for 3 – 5 min.

Precautions:
  • Do not mix by vortexing or vigorous shaking, as this will result in shearing of genomic DNA.
  • Do not allow the lysis reaction to proceed for more than 5 min. Incubation for longer time results in denaturation of supercoiled plasmid DNA. Denatured plasmid will appear as a ghost band in agarose gel analysis of plasmid.
  • Use freshly made lysis solution. Old lysis solution often contains precipitates which can lead to inefficient lysis.

Step 4: Add 150 μl of chilled Neutralization Solution, mix immediately and thoroughly by inverting and rolling the tube 4 – 6 times. Incubate on ice for 3 – 5 min.

Notes:
  • After addition of neutralization solution, a fluffy white material forms and the lysate becomes less viscous. The precipitated material contains genomic DNA, proteins, cell debris and Potassium Dodesyl Sulphate.
Precautions:
  • Use chilled neutralization solution. Precipitation is enhanced by using chilled neutralization solution and incubating on ice.
  • The lysate should be mixed thoroughly to ensure the complete precipitation of SDS in the form of potassium dodecyl sulphate.

Step 5: Centrifuge the tube at maximum speed (14000 rpm) in a microcentrifuge for 10 min at 4°C. Transfer the supernatant containing plasmid promptly in new microcentrifuge tube.

Precautions: While transferring the supernatant, take care that white precipitate should not come along with supernatant. Supernatant should be centrifuged again as mentioned above if supernatant contains any suspended particle.

Step 6 (Optional) : Extract the supernatant with Phenol:Chloroform:isoamylalcohol solution. This step will remove impurities including protein and lipid contamination from the plasmid preparation. Add equal volume of Phenol:Chloroform:Isoamylalcohol (25:24:1) in the supernatant. Mix by vortexing for 10 sec. Centrifuge at maximum speed at 4°C. Transfer the supernatant to fresh microcentrifuge tube.

Precautions:
  • While transferring the supernatant, take care that no traces of phenol come along with supernatant. Traces of phenol is sufficient to inhibits most enzymatic reactions.
  • Phenol and chloroform are toxic. Follow the safety rules while handling phenol.

Step 7: Add equal volume of isopropanol in the supernatant. Mix it by inverting the tube 4 – 6 times. Centrifuge at maximum speed (14,000 rpm) for 30 min at 25°C. Remove the supernatant completely.

Precautions:
  • Incubation for longer time at room temperature or on ice increases plasmid yield, but also causes salt precipitation.
  • While removing the supernatant, care should be taken as isopropanal precipitated plasmid pellet is loosely attached to surface and invisible in most cases. Careless removal of supernatant often results in loss of plasmid pellet.
Step 8: Add 500 μl of 70% ethanol to the pellet. Close the tube and invert several times. Centrifuge at 14000 rpm (maximum speed) for 5 min at 25°C. Remove the supernatant completely.

Tips: To remove the supernatant, one can decant the supernatant after first centrifugation. Remains of liquid will be sticking on the wall of microcentrifuge tube. A second flash spin is sufficient to collect all the liquid at the bottom which can be removed by pipetting. Air dry the pellet for 5 min.

Precautions:
  • Take care with this step, as the pellet sometimes does not adhere tightly to the tube and lost while removing the supernatant.
  • Do not overdry the pellet. Overdried pellet is difficult to dissolve.
  • Remove the traces of ethanol as it may inhibit some enzyme reactions.
Step 9: Dissolve the pellet in 25 μl sterile double distilled water or TE (pH 8.0).

Tips: To dissolve the pellet, one can vortex the solution gently for a brief period ( 2 – 3 times for 5 seconds) and also can incubate at 37° for ∼20 minutes.

Storage

  • Solution can be stored at 4°C for few days. Store at -20°C for years.

Precautions: Don’t thaw the plasmid repeatedly. This can cause reduction of supercoiled form of plasmid.

Applications

  • The isolated plasmid is suitable for most of our cloning experiments. Often the amount of supercoiled plasmid is comparatively less, therefore, is not suitable for transfection experiments.

References:

Birnboim, H.C., and Doly, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7(6), 1513-1523. PMID-388356,

Preparation of 0.5 M EDTA solution

Overview

  • EDTA stands for ethylene diamine tetraacetic acid.
  • It is a polyamino carboxylic acid.
  • It is extensively used in molecular biology experiments as a chelating agent. It sequesters metal ions such as Ca2+ and Fe3+.
  • After being bound by EDTA, metal ions remain in solution but exhibit diminished reactivity.
  • Metal ions are necessary for the action of many enzymes including DNases.
  • EDTA is commercially available as several forms. Disodium salt of EDTA is most commonly used in molecular biology.
  • 0.5 M EDTA solution is used for the preparation of many solutions including TAE, TBE, DNA loading dye, resuspension buffer (isolation of plasmid), Tris-EDTA, Trypsin-EDTA, etc.

Requirement

  • Reagents
    •  EDTA.Na2.2H2O
    • NaOH pallet / 10N NaOH solution (for pH adjustment)
    • Deionized / Milli-Q water
  • Equipment and disposables
    • Measuring cylinder
    • Conical flask / Beaker
    • Magnetic stirrer

Composition:

  • 0.5 M EDTA, pH 8.0 at 25°C

Objective: Preparation of 1000 ml of 0.5 M EDTA solution, pH 8.0 in water from ethylenediaminetetraacetic acid disodium salt, dihydrate (EDTA.Na2.2H2O)

Preparation

Step 1: Weigh out 186.12 grams EDTA.Na2.2H2O (Molecular Weight 372.24). Transfer to 2 L beaker / conical flask. Add 800 ml deionized / Milli-Q water.

Precautions: Do not dissolve in 1000 ml of deionized / Milli-Q water. In most cases, solution volume increases when the large amount of solute dissolves in solvent.

Step 2: While stirring vigorously on a magnetic stirrer, add NaOH pellet or 10N NaOH to adjust the solution pH 8.0.

Tips:
  • ~20 g NaOH pellet is required to adjust the pH 8.0.
  • It is not easy to dissolve EDTA. To dissolve the EDTA completely, solution pH 8.0 is required.

Step 3:  Transfer the solution to autoclavable bottle. Sterilize the solution by autoclaving (20 minutes at 15 lb/sq.in. (psi) from 121-124°C on liquid cycle)

Tips:
  • Depending on the consumption, one can make small aliquots of solution.
Notes:
  • One can sterilize the solution by passing through 0.22 μ filter unit. Filter sterilization removes all suspended particles with size more than 0.22 μ which includes most bacteria their spores but not mycoplasma. However, it does not inactivate enzyme activities (e.g., DNases). Autoclaving inactivates most enzymes except some (e.g., RNases) and kills most microorganisms including mycoplasma.

Storage:

Solution can be stored at 15 – 25 °C (room temperature) for several months.

Follow the table to prepare EDTA solution of different concentration and volume from EDTA.Na2.2H2O (Molecular Weight 372.24).
Conc. / Volume 100 ml 250 ml 500 ml 1000 ml
10 mM 0.37 g 0.93 g 1.86 g 3.72 g
100 mM 3.72 g 9.30 g 18.61 g 37.22 g
0.25 M 9.31 g 23.26 g 46.53 g 93.06 g
0.5 M 18.61 g 46.53 g 93.06 g 186.12 g

 

Tracking dyes

  • Tracking dyes are coloured dyes, which are added to the loading buffer/solution.
  • Loading dye/buffer are used to prepare sample for gel electrophoresis.
  • Tracking dyes serve two purposes:
    • They impart colour to the sample, thus visualizing the sample loading process.
    • Since they are visible by naked eye, the progression of gel electrophoresis can easily be monitored.
  • Tracking dyes should have the following properties in order to be used for gel electrophoresis:
    • They should not interact with the components of sample (DNA or protein).
    • They should move from negative to positive electrode (the direction of electrophoresis). Therefore, they should have net negative charge at pH of electrophoresis buffer.
  • Common tracking dyes used for gel electrophoresis: Bromophenol blue, Xylene cyanol FF, Orange G

Running DNA samples in an agarose gel

Overview:

  • Agarose gel electrophoresis is a very common method of analyzing DNA in most molecular biology laboratories.
  • DNA sample is mixed with DNA loading dye/buffer and placed in wells of agarose gel. Loading buffer helps DNA sample to settled at the bottom of the wells.
  • Since DNA is negatively charged due to its phosphate group, it moves towards positive pole under the electric field.
  • Often a small amount of ethidium bromide is added in the agarose gel to visualize DNA. Alternatively gel can be placed in a ethidium bromide containing solution to visualize DNA after electrophoresis.
  • Movement of DNA is primarily controlled by size and form (conformation) of DNA, agarose concentration in the gel and strength of electric field.
  • Higher percentage (2%) agarose gel is used to resolve small size DNA fragments whereas lower percentage agarose gel is used to resolve large DNA fragments.
  • DNA fragments lower than 50 bp are separated by polyacrylamide gel electrophoresis. Larger DNA fragments (more than 30 kB) are separated by pulse field gel electrophoresis.

Requirements:

Reagents:

  • Agarose gel (see preparation process)
  • Ethidium bromide solution (10 mg/ml in water)
  • Electrophoresis buffer (TAE or TBE Buffer)

Tips: Use the same electrophoresis buffer which was used to prepare agarose gel (e.g., Use TAE if the agarose gel was prepared in TAE).

Equipment and disposables:

  • Micropipettes and tips
  • Gloves
  • Electrophoresis Power supply
  • Electrophoresis apparatus
  • UV transilluminator or Gel documentation

Protocol:

Step 1: Place the agarose gel with casting tray in the electrophoresis tank. Since DNA moves from negative to positive electrode, the side where the wells are, should be towards the negative electrode.

Notes:

  • Different kinds of gel casting apparatus are available. In some cases, you need to seal the edges with the tape, whereas, in others you need to just place casting tray into a cassette.

Precautions:

  • If casting tray is sealed with tape, remove the tape from both sides. Tape will not allow the passage of electric current through gel.
  • Don’t remove the gel from the casting tray. This may cause damage to the wells.

Step 2: Fill the electrophoresis tank with electrophoresis buffer.

Tips:

  • We recommend you to add ethidium bromide to a final concentration of 0.5 μg/ml in the electrophoresis buffer if the the agarose gel contains ethidium bromide. For example, add 50 µl ethidium bromide (stock conc 10 mg/ml)  in 1000 ml electrophoresis buffer.
  • As the electrophoresis progress, positively charged ethidium bromide moves towards negative electrode, resulting in depletion of ethidium bromide from the rear end of the agarose gel. This results in appearance of dark zone in the agarose gel when you analyze gel under uv trans-illuminator.
  • Use the same electrophoresis buffer which was used to prepare agarose gel (e.g., Use TAE if the agarose gel was prepared in TAE)
  • If you are using TAE electrophoresis buffer, pour 1X TAE buffer in the tank. For TBE, use 0.5X TBE electrophoresis buffer.

Precautions:

  1. Agarose gel should be submerged in buffer, but don’t fill electrophoresis buffer too much. Too much electrophoresis buffer over the agarose gel can cause slow run and distorted DNA band.
  2. Ethidium bromide is carcinogenic. Use appropriate safety measures (use latex gloves, wear lab coat) to avoid any harm.

Step 3: Carefully remove the comb from the gel without causing damage to the wells.

Notes:

Comb can be removed even before placing the gel in the electrophoresis buffer, but occasionally, this can cause collapsing of wells. It is much more safer to remove the comb after submerging the gel in electrophoresis buffer. Electrophoresis buffer moves inside the wells as the comb is removed, thus protecting the wells from collapsing.

Precautions: Wells should not be damaged. Often wells are damaged at the bottom that may go uncheck, causing the leakage and loss of samples. If the samples are precious, check the well by loading a small amount of 1X loading dye in the well.

Step 4: Prepare the DNA sample by mixing 5 volume of DNA solution with 1 volume of 6X DNA loading dye (e.g., 5 μl DNA solution and 1 μl DNA loading dye). Mix it and load on the wells of agarose gel carefully without spilling the sample into the adjacent wells.

Tips:

  • If DNA sample need to be diluted, one can use water or more conveniently the electrophoresis buffer from the tank.
  • It is very convenient and economical to mix the DNA solution with loading dye on the parafilm instead of using microcentrifuge tubes. To mix the sample with dye, one can place the required amount of dye on the parafilm. Now add DNA solution and mix it by few rounds (4 – 5 times) of pipetting.

Precautions:

  • Do not destroy well while sample loading.
  • While pipetting DNA sample, avoid air-bubbles. Air-bubbles can cause spillage of DNA sample into adjacent wells.

Step 5: Connect the electrophoretic apparatus to power supply and start the gel run.

Notes:

  • Place the lid on the gel box, and connect electrodes with power supply using electric wires, supplied with the electrophoresis apparatus. Set the current maximum and voltage 70 – 100 volt. Turn the power supply on.
  • The distance between electrodes in electrophoretic apparatus determines maximum voltage. Voltage should not exceed 5 volts / cm. Since most commonly used electrophoresis devices have distance 14 – 20 cm between electrodes, 70 – 100 volt is default voltage.

Tips:

By looking air-bubbles from the electrodes (negative electrode), one can ensure that the electric supply is functional.

Precautions:

  • Always place the lid of electrophoretic apparatus to avoid electric shock.
  • Make sure that connections [to positive (red in colour) and negative poles (blue in colour)] to power pack is proper. If it is reverse, sample will run on the opposite direction and will come out from the gel. The direction of the run can be monitored by observing the movement of the loading dye. (it will run in the same direction as the DNA).

Step 6: Run the agarose until the purple dye (represents Bromophenol blue) approaches the end of the gel or 3/4 of the gel. Turn off the power supply and disconnect the wires from the power pack.

Notes: Depending on the DNA size and resolution of DNA fragments, one has to decide the gel run time.

Step 7: Once the gel run over, turn off the power supply and disconnect the wires. Take out the gel from the electrophoresis chamber and analyze under UV transilluminator or Gel documentation system.

Tips: Immediately analyze the gel for DNA just after you disconnect electric supply. Storing gel in electrophoresis buffer with run can cause loss of resolution due to diffusion of DNA band.

Precautions:

  • Use hand gloves and follow safety rules as the gel contains ethidium bromide.
  • Use tray to take out gel. Some casting trays allow direct visualization of gel as they are permeable to UV light but are costlier. If casting tray is impermeable to UV light, take out the gel and analyze under UV light.
  • Uv is harmful. Use UV Safety Goggles and shield to protect yourself from direct exposure to uv rays.

 

 

Preparation of agarose gel for DNA analysis

Overview

  • Agarose gel electrophoresis is a very common method in all molecular biology laboratories. It has many applications including analysis of DNA (size analysis, detection of DNA in a sample, separation and purification of DNA fragments etc.).
  • Agarose gel is a gelatin like slab, which contains small wells. It is prepared by melting agarose in a suitable electrophoresis buffer.
  • Percentage of agarose in the gel decides the pore size through which DNA moves. Electrophoresis buffer facilitates the passage of electric current through gel.
  • Melted agarose is poured in a specialized tray (casting tray), which control the size of the gel (the amount of agarose solution is dependent primarily on size of the casting tray). Comb is used to create wells in agarose. gel. Well facilitates the agarose gel to keep the DNA sample.
  • Solidified agarose gel is submerged into electrophoresis buffer. Since DNA is negatively charged and moved towards the positive electrode, side of the gel with wells is always placed towards the negative electrode. A electrophoresis power supply apparatus is used to maintain electric field in the electrophoresis tank.
  • Movement of DNA is primarily controlled by size and form (conformation) of DNA, agarose concentration in the gel and strength of electric field. Often a small amount of ethidium bromide is added in the agarose gel to visualize DNA. Alternatively gel can be placed in a ethidium bromide containing solution to visualize DNA after electrophoresis.

Requirements

Reagents:

  • Agarose
  • Ethidium bromide solution (10 mg/ml in water)
  • Electrophoresis buffer (TAE or TBE Buffer)

Equipment and disposables:

  • Gel casting tray and combs
  • Micropipettes and tips
  • Gloves

    Objective

Preparation a 0.8 % agarose gel (Gel size – 8 cm X 10 cm X 0.5 cm) in TAE buffer

Note : Depending on the need, one can prepare various percentage of agarose gel (See table). Here we have taken an example to describe the preparation process clearly.


Preparation:

Step 1: Dissolve the agarose in electrophoresis buffer

  • Weigh out 0.4 gm agarose in a conical flask. Add 50 ml 1X TAE buffer in this. Suspend the agarose by swirling the flask. Wait for 1 – 2 min to allow hydration of agarose particles.
  • Melt the agarose in microwave or hot plate until the solution becomes clear.

Tips

  1. To make 0.8 % agarose gel of size 8 cm (width) X 10 cm (length) X 0.5 cm (thickness), 50 ml solution is sufficient.
  2. The total gel volume varies depending on the size of the casting tray. To decide how much volume of agarose solution is required for a casting tray, one can use water to get an idea about the volume of agarose solution required.

Precautions:

  1. The size of the flask should be atleast 5 times of the volume of the agarose solution to be prepared. While heating, solution boils and comes outside. So if the size of the flask is not appropriate, one may lose some part of the solution.
  2. If microwave or hot plate is used to melt the agarose, heat the solution for several short intervals. Avoid continuous boiling of the solution as it may boil out of the flask. Alternatively one can use boiling water bath to melt the agarose. It may take longer time.
  3. Make sure that melted agarose solution appear clear and transparent, devoid of any suspended particles of agarose. If there are some suspended particles, melt it more.
  4. Sometimes there is a significant loss of water, which depends on the melting procedure. Measure the total volume of melted agarose solution, and if the volume is significantly less, make up the loss by adding deionized water (Do not add buffer). The best way is to place the flask to weighing balance just before starting the melting process and set the balance zero. After melting agarose, again place the flask containing melting agarose solution on the balance. You will see negative reading, which will be equal to the loss of water during the process of melting agarose. Adjust this negative value to 0 by adding deionized water to the melted agarose solution.

Step 2: Add Ethidium bromide to agarose solution (optional)

  • Cool the solution until the temperature reaches 50-55°C. Swirl the flask occasionally to cool solution evenly. One can prepare the casting tray during this time.
  • Add 2.5 μl ethidium bromide in the solution. Mix by gentle swirling. Avoid air bubble formation.

Precautions:

  1. Ethidium bromide is carcinogenic. Use appropriate safety measures (use latex gloves, wear lab coat) to avoid any harm.
  2. Do not add Ethidium bromide when the solution is very hot.

Step 3: Pour the melted agarose solution into a casting tray

  • Prepare the casting tray by sealing both the open ends of tray with tape. Pour the melted agarose solution into a casting tray. Insert the comb at appropriate place.
  • Wait until agarose is solidified completely. Solidified agarose gel will appear milky white.

Tips:

  1. One can place the comb before pouring the agarose solution into the casting tray. 2: Agarose gel can be stored for several days. To store the agarose gel, we recommend not to remove comb and tape. Dip the agarose gel in the TBE buffer so that it contains moisture. Seal the gel in a plastic wrap. Store in the cold room (4°C). In case gel becomes dry, allow it to rehydrate with in the electrophoresis (TAE or TBE) buffer for few minutes. Before starting electrophoresis, let it come to room temperature.
  1. Precautions:
    1. Avoid air bubble. Remove air bubble with the help of pipette tip.
    2. While inserting the comb, take care that teeth of comb should not touch the bottom of casting tray. If it touches, Well will be like a hole. In this case, sample will come out from the well.

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

Overview

  • This method uses 40% sucrose solution instead of sucrose powder.
  • This method is convenient and easy, and does not involve adjustment of volume.

Requirements

Reagents:

  • Bromophenol blue
  • Xylene cyanol FF
  • 40% (W/V) Sucrose solution
  • Deionized / Milli-Q water

Equipment and disposables:

  • Measuring cylinder
  • Conical flask / Beaker / 15 ml screw-capped tube
  • Rotator

Composition

  • 0.25% (W/V) bromophenol blue
  • 0.25% (W/V) xylene cyanol FF
  • 40% (W/V) sucrose

Objective

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


Preparation:

  • To prepare 10 ml of 6X DNA loading dye, weigh out 25 mg bromophenol blue and 25 mg xylene cyanol FF. Transfer it to screw-capped tube (15 ml with millilitre marks) / beaker / conical flask. Add 10 ml of 40% sucrose solution. Mix until all ingredients are dissolved completely.

Note: This method is simple and convenient as it does not involve readjustment of solution volume. There will not be any significant change in the volume of 40% sucrose solution when you dissolve small amount of  bromophenol blue and xylene cyanol FF in it.

Tips: We recommend to use disposable nuclease nuclease-free (DNase- and RNase-free) 15 ml screw-capped tube with millilitre marks. Transfer all the contents in it. Add 10 ml 40% sucrose solution and mix the content by inverting the tube number of times or using rotator.


Storage

  • Store the solution at -20°C for long time. Solution can be stored at 2 – 8°C for few weeks.

Tips: It is recommended to store the solutions in small aliquots (1 ml).


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 cynol FF 12.5 mg 25 mg 62.5 mg 125 mg 250 mg
40% Sucrose solution 5 ml 10 ml 25 ml 50 ml 100 ml

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

Overview

  • DNA sample is mixed with DNA loading dye prior to loading into the wells of agarose gel.
  • A DNA loading dye must contain at least one dye (orange G, bromophenol blue, xylene cyanol FF or bromocresol green) and a high density reagent (glycerol, sucrose or Ficoll 400).
  • Dyes help to track the progression of gel electrophoresis and sample loading process in the well.
  • Two dyes containing DNA loading dye is very common for DNA gel electrophoresis. The most common dyes are bromophenol blue and xylene cyanol FF.
  • Bromophenol blue migrates fast in the agarose gel and corresponds to the migration of 300 – 500 bp long DNA fragment in 1% agarose gel.
  • Xylene cyanol FF migrates comparatively slow and corresponds to the migration of 4000 – 5000 bp long DNA fragment in 1% agarose gel.
  • A 6X DNA loading dye can have dye concentration ranging from 0.03% to 0.50% (W/V). High concentration of dye provides very good contrast colour, which is easy to monitor upon electrophoresis progression. However, high dye concentration masks the co-migrating DNA fragments, causing wrong analysis of co-migrating DNA bands (e.g., densitometric analysis). Low concentration of dye is preferred when DNA sample is expected to contain co-migrating DNA fragment(s). However, low concentration of dye causes a compromise in the visibility of migrating dye band, which sometime disappear after a long electrophoresis run.
  • Sucrose is added to provide high density to solution. Due to high density, sample settle at the bottom of the well. It also helps DNA sample to be confined in the well without diffusing out.

Requirement

Reagents:

  • Bromophenol blue
  • Xylene cyanol FF
  • Sucrose
  • Deionized / Milli-Q water

Equipments and disposables:

  • Measuring cylinder
  • Conical flask / Beaker / 15 ml screw-capped tube
  • Rotator

Composition

  • 0.25% (W/V) bromophenol blue
  • 0.25% (W/V) xylene cyanol FF
  • 40% (W/V) sucrose

Objective

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


Preparation:

  • To prepare 10 ml of 6X DNA loading dye, weigh out 25 mg bromophenol blue, 25 mg xylene cyanol FF and 4 gm Sucrose. Transfer them to 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 nuclease-free (DNase- and RNase-free) 15 ml screw-capped tube with millilitre marks. Transfer all the contents in it. Add 7 ml deionized / Milli-Q water and mix the content by inverting the tube number of times or using rotator. Once the content is mixed, adjust the volume to 10 ml. 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 is viscous and contains dye.

Precautions:

  1. Do not dissolve in 10 ml of deionized / Milli-Q water. In most cases, solution volume increases when the large amount of solute dissolves in solvent.
  2. Use nuclease-free, autoclaved deionized / Milli-Q water and glasswares.
  • Adjust the volume to 10 ml with deionized / Milli-Q water. Mix it again.

     

Storage

  • Store the solution at -20°C for long time. Solution can be stored at 2 – 8°C for few weeks.

Tips: It is recommended to store the solutions in small aliquots (1 ml).


 

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 cynol FF 12.5 mg 25 mg 62.5 mg 125 mg 250 mg
Sucrose 2 gm 4 gm 10 gm 20 gm 40 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