Preparation of solutions for Molecular Biology experiments.

1. Tris-Acetate-EDTA (TAE) Buffer Preparation

TAE buffer is frequently used for agarose gel electrophoresis when analyzing large DNA fragments.

Components for 1 Liter of 50x TAE Stock Solution

 Tris Base: 242 g

 Glacial Acetic Acid: 57.1 mL
 0.5 M EDTA (pH 8.0): 100 mL

Procedure

1. Dissolve Tris Base:

o Weigh 242 g of Tris base and place it into a large beaker or container.
o Add about 600–700 mL of distilled water and stir until the Tris is completely dissolved.

2. Add Glacial Acetic Acid:

o Carefully measure 57.1 mL of glacial acetic acid and add it to the Tris solution while stirring.

3. Add EDTA:
o Measure 100 mL of 0.5 M EDTA (pH 8.0) and add to the solution.

4. Adjust the Volume:

o Add distilled water to bring the final volume to 1 liter.

5. Storage:
o Store the 50x TAE stock solution at room temperature. It is stable for several months.

To Prepare 1x TAE Working Solution

 Dilution: To make a 1x TAE buffer from the 50x stock, dilute 20 mL of the 50x TAE with 980 mL of distilled water.
 Final Concentration: The 1x TAE buffer will have a final concentration of 40 mM Tris-acetate and 1 mM EDTA (pH ~8.0).

2. Tris-Borate-EDTA (TBE) Buffer Preparation

TBE buffer is more commonly used when sharper resolution is required, especially for smaller DNA fragments, and it provides better
buffering capacity for extended electrophoresis runs.

Components for 1 Liter of 10x TBE Stock Solution

 Tris Base: 108 g

 Boric Acid: 55 g
 0.5 M EDTA (pH 8.0): 40 mL

Procedure

1. Dissolve Tris Base and Boric Acid:

o Weigh 108 g of Tris base and 55 g of boric acid, and add them to a large beaker or container.
o Add about 600–700 mL of distilled water and stir until fully dissolved.

2. Add EDTA:
 o Measure 40 mL of 0.5 M EDTA (pH 8.0) and add to the Tris-boric acid solution.

3. Adjust the Volume:

o Add distilled water to bring the total volume to 1 liter.

4. Storage:
o Store the 10x TBE stock solution at room temperature. It is stable for several months.

To Prepare 1x TBE Working Solution

 Dilution: To make a 1x TBE solution, dilute 100 mL of the 10x TBE stock with 900 mL of distilled water.
 Final Concentration: The 1x TBE buffer will have a final concentration of 89 mM Tris, 89 mM boric acid, and 2 mM
EDTA (pH ~8.3).

Key Considerations

 pH Adjustment: Usually not required, as the pH should be correct if the solutions are prepared accurately.
 Autoclaving: Not recommended for TAE or TBE as it may alter the buffering properties. These buffers are generally stable
and can be stored without sterilization.
 Storage: Store at room temperature, protected from light.

1. Tris-HCl Buffer Preparation

Tris-HCl is commonly used in many molecular biology protocols due to its buffering capacity in the pH range of 7.0–9.0.

For 1 Liter of 1 M Tris-HCl (pH 7.4)

 Tris Base: 121.1 g

 Hydrochloric Acid (HCl): To adjust the pH (typically 5–10 mL of concentrated HCl, but varies)
 Distilled Water: To bring the volume to 1 liter

Procedure

1. Dissolve Tris Base:

o Weigh 121.1 g of Tris base and place it into a beaker.
o Add approximately 800 mL of distilled water and stir until the Tris is completely dissolved.

2. Adjust the pH:

o Using a pH meter, slowly add concentrated HCl (dropwise or using a pipette) while stirring to bring the solution to pH 7.4.
o Note that the pH will stabilize after a few minutes of stirring, so adjust gradually to avoid overshooting.

3. Bring to Final Volume:

o After reaching the desired pH, add distilled water to bring the total volume to 1 liter.

4. Storage:
o Store at room temperature or 4°C. This solution is stable for several months.

Dilution for Working Solutions

 Example: To make a 0.1 M Tris-HCl solution, dilute the 1 M stock solution by 10x using distilled water.

2. Phosphate-Buffered Saline (PBS) Preparation

PBS is widely used in cell culture and molecular biology as it maintains osmolarity and pH in physiological ranges.
Components for 1 Liter of 1x PBS (pH 7.4)

 NaCl (Sodium Chloride): 8 g

 KCl (Potassium Chloride): 0.2 g
 Na₂HPO₄ (Disodium Phosphate): 1.44 g
 KH₂PO₄ (Monopotassium Phosphate): 0.24 g
 Distilled Water: To bring the volume to 1 liter

Procedure

1. Dissolve Salts:
o Add 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na₂HPO₄, and 0.24 g of KH₂PO₄ into a beaker.
o Add about 800–900 mL of distilled water and stir until all salts are dissolved.

2. Adjust pH (if needed):

o Check the pH, which should be around 7.4. Usually, no adjustment is needed. However, if necessary, adjust the pH with a few
drops of 1 M HCl or 1 M NaOH.

3. Bring to Final Volume:

o Add distilled water to bring the total volume to 1 liter.

4. Sterilization:
o Sterilize by autoclaving (121°C for 15–20 minutes) or by filtration through a 0.22 µm filter if required for sterile applications.

5. Storage:
o Store the 1x PBS solution at room temperature or 4°C. PBS is stable for several months.

10x PBS Stock Solution Preparation

 To prepare a 10x PBS stock solution (concentrated), multiply each salt amount by 10 and follow the same preparation steps.
 For use, dilute the 10x stock solution 1:10 with distilled water to create 1x PBS.

1. DNA Extraction Solutions

DNA extraction typically involves several solutions to lyse cells, protect DNA from degradation, and precipitate it for purification.

A. Lysis Buffer

 Purpose: Breaks down cell membranes and releases DNA into solution.
 Common Components:
o Tris-HCl (10 mM): Maintains a stable pH, typically around pH 8.0.
o EDTA (1-2 mM): Chelates divalent metal ions, protecting DNA from nucleases.
o SDS (Sodium Dodecyl Sulfate, 0.5%): Detergent that lyses cells and denatures proteins.
o NaCl (100-200 mM): Provides ionic strength, helping to stabilize DNA.

Preparation for 100 mL of DNA Lysis Buffer

 Tris Base (10 mM): Dissolve 0.121 g in distilled water.

 EDTA (1 mM): Dissolve 0.037 g in distilled water.
 NaCl (100 mM): Dissolve 0.584 g in distilled water.
 SDS (0.5%): Add 0.5 g of SDS.
 Adjust pH to 8.0 with HCl if necessary.
 Bring to Final Volume: Add distilled water to reach 100 mL.
 Storage: Store at room temperature or 4°C for short-term use.

B. Proteinase K Solution (Optional)

 Purpose: Digests proteins that may be bound to DNA.

 Preparation:
o Dissolve proteinase K to a final concentration of 20 mg/mL in distilled water or buffer.
o Storage: Store at -20°C to maintain activity.
C. DNA Precipitation Solution (Ethanol/Isopropanol)

 Purpose: Precipitates DNA out of solution.

 Preparation:
o For ethanol precipitation, use cold 100% ethanol.
o For isopropanol precipitation, use cold 100% isopropanol.
o Procedure: After adding ethanol or isopropanol to the DNA solution, centrifuge at high speed to pellet the DNA.

D. TE Buffer (Tris-EDTA) for DNA Storage

 Purpose: Protects DNA during storage.

 Components:
o 10 mM Tris-HCl (pH 8.0)
o 1 mM EDTA
 Preparation for 100 mL of TE Buffer:
o Tris Base (10 mM): Dissolve 0.121 g in distilled water.
o EDTA (1 mM): Dissolve 0.037 g in distilled water.
o Adjust pH to 8.0 with HCl.
o Bring to final volume with distilled water to make 100 mL.
 Storage: Store at room temperature or 4°C.

2. RNA Extraction Solutions

RNA extraction requires solutions that stabilize RNA and prevent degradation, as RNA is more sensitive to RNases than DNA.

A. RNA Lysis Buffer (e.g., Guanidinium Thiocyanate Buffer)

 Purpose: Lysing cells and denaturing proteins to release RNA and protect it from RNase activity.
 Common Components:
o Guanidinium Thiocyanate (4 M): A strong chaotropic agent that denatures proteins and RNases.
o Sodium Citrate (0.1 M, pH 7.0): Stabilizes pH.
o Sarkosyl (0.5%): A detergent that assists in cell lysis and protein denaturation.
o 2-Mercaptoethanol (optional, 0.1-0.2%): Reducing agent to inactivate RNases.

Preparation for 100 mL of RNA Lysis Buffer

 Guanidinium Thiocyanate (4 M): Dissolve 47.3 g in distilled water.

 Sodium Citrate (0.1 M, pH 7.0): Dissolve 2.94 g in distilled water.
 Sarkosyl (0.5%): Add 0.5 g.
 2-Mercaptoethanol: Add 0.1-0.2 mL just before use (not stable long-term).
 Adjust pH to 7.0 with HCl if necessary.
 Bring to Final Volume: Add distilled water to reach 100 mL.
 Storage: Store at room temperature, protect from light.

B. Phenol:Chloroform
Alcohol (PCI) Solution (25:24:1)

 Purpose: Organic solvent mixture used to separate RNA from proteins and other contaminants.
 Preparation:
o 25 mL of Phenol (equilibrated to pH 4.5–5.0 for RNA).
o 24 mL of Chloroform.
o 1 mL of Isoamyl Alcohol (prevents foaming).
o Combine in a light-protected container, and shake well.
 Storage: Store in dark glass at 4°C and handle in a fume hood due to toxicity.

C. RNA Precipitation Solution (Isopropanol or Ethanol)

 Purpose: Precipitates RNA after extraction.

 Preparation:
 o Use 100% isopropanol (recommended for RNA) or 75% ethanol.
o Procedure: After extraction, add isopropanol or ethanol to the RNA solution, mix well, and centrifuge to pellet the RNA.

D. DEPC-Treated Water for RNA Storage

 Purpose: DEPC (Diethyl Pyrocarbonate)-treated water inactivates RNases.

 Preparation:
o Add 0.1% DEPC to distilled water.
o Stir for 1 hour at room temperature.
o Autoclave the solution to inactivate the DEPC.
 Storage: Store at room temperature or 4°C. Use this water to dilute or resuspend RNA, ensuring it remains RNase-free.

Competent Cell Preparation with CaCl₂

Overview

 Purpose: To make bacterial cells (e.g., E. coli) more permeable to DNA by treating them with CaCl₂ and making them cold-sensitive.
 Method: Cold CaCl₂ treatment followed by a brief incubation on ice helps stabilize the cell membrane and prepares cells for DNA uptake.

Materials Needed

 Calcium Chloride (CaCl₂)

 Glycerol (optional, for long-term storage of competent cells)
 Distilled Water: Preferably sterile
 Sterile Centrifuge Tubes: 15 mL or 50 mL tubes
 Ice: Essential to keep cells cold throughout the preparation
 Bacterial Culture: Typically E. coli grown to mid-log phase

1. Preparation of 0.1 M CaCl₂ Solution

 Components:
o Calcium Chloride (CaCl₂): 1.47 g
o Distilled Water: Up to 100 mL final volume

 Procedure:

o Weigh and Dissolve: Measure 1.47 g of CaCl₂ and dissolve it in about 80 mL of distilled water.
o Bring to Final Volume: Add distilled water to bring the total volume to 100 mL.
o Sterilize the Solution: Filter sterilize through a 0.22 µm filter or autoclave the solution.
o Chill on Ice: Place the CaCl₂ solution on ice before use, as it needs to be very cold for competent cell preparation.

 Storage: Store the 0.1 M CaCl₂ solution at 4°C for short-term use. It remains stable for a few weeks when kept cold and sterile.

2. Preparation of 0.1 M CaCl₂ with 15% Glycerol (for Cell Storage)

 Components:
o Calcium Chloride (CaCl₂): 1.47 g
o Glycerol: 15 mL
o Distilled Water: Up to 100 mL final volume
 Procedure:

o Weigh and Dissolve CaCl₂: Measure 1.47 g of CaCl₂ and dissolve in about 50 mL of distilled water.
o Add Glycerol: Slowly add 15 mL of glycerol, mixing thoroughly.
o Bring to Final Volume: Add distilled water to reach a total volume of 100 mL.
 o Sterilize: Filter sterilize the solution through a 0.22 µm filter.
o Chill on Ice: Keep the solution on ice before use.

 Storage: Store at 4°C. The addition of glycerol allows you to freeze competent cells for long-term storage.

3. Competent Cell Preparation Protocol

Materials Needed

 Fresh overnight culture of E. coli grown in LB or a suitable medium.

 Pre-chilled 0.1 M CaCl₂ solution (or 0.1 M CaCl₂ with 15% glycerol for freezing).
 Ice-cold sterile centrifuge tubes (15 mL or 50 mL).

Procedure

1. Inoculate and Grow Bacterial Culture:

o Inoculate E. coli in LB broth and grow overnight at 37°C with shaking.
o The next day, dilute the overnight culture 1:100 into fresh LB broth and continue shaking at 37°C until the culture reaches an
OD600 of 0.4–0.6 (mid-log phase).

2. Harvest Cells:
o Place the culture on ice for 10 minutes before harvesting.
o Transfer the culture to ice-cold centrifuge tubes and centrifuge at 4°C, 3000 x g, for 10 minutes to pellet the cells.

3. Wash Cells with Cold CaCl₂:

o Discard the supernatant carefully and resuspend the cell pellet in an equal volume of ice-cold 0.1 M CaCl₂ (e.g., 10 mL of CaCl₂
solution for each 10 mL of culture).
o Incubate the cells on ice for 10–30 minutes to increase their competency.

4. Second Centrifugation:
o Centrifuge the cells again at 4°C, 3000 x g, for 10 minutes.
o Carefully discard the supernatant.

5. Resuspend Cells in CaCl₂ with Glycerol (Optional):

o For long-term storage, resuspend the cell pellet in ice-cold 0.1 M CaCl₂ with 15% glycerol. Use 0.5–1 mL for each 50 mL of
initial culture.
o For immediate use, resuspend cells in ice-cold 0.1 M CaCl₂ without glycerol.

6. Aliquot and Store Cells:

o Aliquot the competent cells into sterile, pre-chilled tubes (50–100 µL per tube).
o If storing long-term, flash-freeze the cells in liquid nitrogen or a -80°C freezer. They can be stored for up to several months at -
80°C.

Agarose gel electrophoresis of genomic DNA Materials Required

 Agarose: For gel preparation, typically 0.8–1% agarose for genomic DNA.
 Electrophoresis Buffer: Commonly 1x TAE (Tris-Acetate-EDTA) or 1x TBE (Tris-Borate-EDTA).
 DNA Stain: Ethidium bromide, SYBR Safe, or GelRed to visualize DNA (handle with care if using ethidium bromide).
 DNA Loading Dye: Typically 6x loading dye containing glycerol or Ficoll and tracking dyes (e.g., bromophenol blue, xylene cyanol).
 DNA Ladder: A molecular weight marker for reference.
 Electrophoresis Tank and Power Supply: To run the gel.
 UV Transilluminator or Gel Documentation System: For DNA visualization.

1. Preparation of the Gel

A. Determine Gel Concentration

 Use a 0.8–1% agarose gel for genomic DNA. Lower percentage gels (e.g., 0.8%) allow better separation of large DNA fragments.

B. Gel Preparation

1. Calculate and Weigh Agarose:

o For a 1% gel in a 100 mL volume, weigh 1 g of agarose.

2. Dissolve Agarose:
o Add the agarose powder to 100 mL of 1x TAE or 1x TBE buffer in a heat-resistant flask.
o Heat the mixture in a microwave or hot water bath until the agarose is completely dissolved. Avoid boiling over.

3. Cool and Add DNA Stain (Optional):

o Allow the agarose solution to cool to about 60°C (warm to touch).
o If using a non-toxic stain (e.g., SYBR Safe), add it to the gel at the recommended concentration.
o Ethidium bromide can also be added at 0.5 µg/mL if not staining the gel post-run (use caution as it’s a mutagen).

4. Pour the Gel:

o Place a gel tray in the electrophoresis tank with the comb inserted to form wells.
o Pour the warm agarose solution into the tray, ensuring no bubbles form in the wells.
o Allow the gel to solidify for 20–30 minutes at room temperature.

2. Preparation of Samples

1. Prepare DNA Sample with Loading Dye:

o Mix genomic DNA with 6x loading dye at a 1:5 ratio. For example, add 2 µL of 6x dye to 10 µL of DNA sample.
o Loading dye contains glycerol or Ficoll to help the sample sink into the wells and tracking dyes to monitor the progress.

2. Prepare DNA Ladder:

o Dilute an appropriate amount of DNA ladder (molecular weight marker) in loading dye. This ladder will serve as a reference for
estimating DNA fragment sizes.

3. Loading the Gel

1. Submerge the Gel:

o Once the gel has solidified, remove the comb carefully.
o Place the gel tray in the electrophoresis tank and add enough 1x TAE or 1x TBE buffer to just cover the gel (about 2–3 mm
above the gel surface).

2. Load Samples into Wells:

o Using a micropipette, carefully load the prepared DNA samples and DNA ladder into separate wells.
o Be careful not to puncture the gel while pipetting, and ensure the samples are evenly loaded into the wells.

4. Running the Gel

1. Set Up the Power Supply:

o Connect the electrophoresis tank to a power supply, ensuring the wells (negative end) are oriented toward the cathode (black
electrode).
o DNA is negatively charged and will migrate toward the positive anode (red electrode).

2. Run the Gel:

o Set the power supply to 80–120 volts, depending on gel size. A standard gel typically runs at 100 volts.
o Run the gel for 1–1.5 hours, or until the tracking dye (e.g., bromophenol blue) has migrated about 3/4 of the gel length.
5. Staining and Visualization (if not pre-stained)

 Ethidium Bromide Staining (if added post-run):

o Prepare an ethidium bromide staining solution (0.5 µg/mL) in water or buffer.
o Soak the gel in the staining solution for 10–15 minutes, followed by a 5–10 minute rinse in water to reduce background
fluorescence.

 Alternative Stains (e.g., SYBR Safe):

o Follow the manufacturer’s instructions for staining with safer alternatives like SYBR Safe or GelRed.

6. Visualization of DNA

1. Place Gel on UV Transilluminator:

o Carefully place the gel on a UV transilluminator or gel documentation system.
o Caution: Use UV protective goggles and minimize exposure time if using ethidium bromide.

2. Analyze the Results:

o Genomic DNA typically appears as a large, high molecular weight band near the top of the gel.
o Compare the DNA bands to the DNA ladder to estimate size.
o Capture an image if using a gel documentation system.

Troubleshooting Tips

 Smearing: Smearing in the lane may indicate degraded DNA. Ensure samples are stored properly and that the DNA is not degraded.
 Poor Resolution: If bands are not sharp, try running the gel at a lower voltage or using fresh buffer.
 Faint Bands: Use more DNA in the sample, ensure the stain is at the correct concentration, or increase staining time.
 Running Direction: Confirm the DNA wells are near the negative electrode (cathode), as DNA migrates toward the positive electrode.

Quick Reference Table

Step Reagents/Equipment Details

Gel Preparation Agarose, 1x TAE/TBE buffer 0.8–1% gel, pre-warmed to 60°C
Sample Preparation DNA, loading dye Mix DNA with 6x loading dye
Loading the Gel Gel tray, pipette Load DNA and ladder in wells
Running the Gel Electrophoresis tank, power supply 100 volts, 1–1.5 hours
Visualization UV transilluminator Observe bands under UV light
 Separation of nucleotide bases by paper chromatography

Materials Required

 Chromatography Paper: Whatman filter paper (No. 1 or similar)

 Solvent (Mobile Phase): A common solvent for nucleotide base separation is a mixture of butanol, acetic acid, and water (typically in a 4:1:5 ratio).
 Nucleotide Base Standards: Solutions of adenine, thymine, guanine, cytosine, and uracil at a known concentration.
 Capillary Tubes or Micropipette: For spotting the nucleotide solutions onto the paper.
 Developing Chamber: A glass jar with a lid, large enough to hold the chromatography paper.
 Pencil and Ruler: For marking the paper.
 UV Light Source: For visualizing separated nucleotide bases (optional).
 Ninhydrin or other Staining Solution: To visualize nucleotide bases after separation if UV light is unavailable.

1. Preparation of the Solvent (Mobile Phase)

 Solvent Composition: Prepare a mixture of butanol, acetic acid, and water in a 4:1:5 ratio.
o For example, mix 40 mL of butanol, 10 mL of acetic acid, and 50 mL of distilled water.
 Mix Thoroughly: Ensure the solvent is well-mixed and homogeneous.
 Pour into Developing Chamber: Add the solvent mixture to the developing chamber (e.g., a large glass jar) to a depth of about 1 cm.
 Pre-saturate the Chamber: Close the chamber and let it sit for 10–15 minutes to allow the solvent vapor to saturate the chamber.

2. Preparing the Chromatography Paper

1. Cut the Paper:

o Cut a piece of chromatography paper to fit the developing chamber, typically a strip around 15–20 cm long and 5–7 cm wide.

2. Draw the Baseline:

o Using a pencil, draw a horizontal line about 2 cm from the bottom of the paper. This will be the baseline for spotting the samples.
o Mark the Sample Spots: Along the baseline, lightly mark points where each nucleotide base will be spotted (around 1 cm apart).

3. Spot the Samples:

o Use a capillary tube or micropipette to apply a small amount (1–2 µL) of each nucleotide solution onto the designated spots on
the baseline.
o Allow the spots to dry, then reapply several times to concentrate each spot, ensuring the samples are compact for better
resolution.

3. Developing the Chromatogram

1. Place the Paper in the Chamber:

o Roll the chromatography paper into a cylinder, ensuring it doesn’t touch the sides of the chamber, or hang it straight down into
the solvent.
o Ensure that the baseline is above the level of the solvent (approximately 1 cm above the solvent surface).

2. Develop the Chromatogram:

o Close the chamber lid to prevent evaporation of the solvent.
o Allow the solvent to rise up the paper by capillary action, carrying the nucleotide bases along with it.
o The bases will move at different rates based on their affinity for the paper (stationary phase) and their solubility in the solvent
(mobile phase), causing them to separate.
o Allow the solvent front to travel up the paper until it is about 1–2 cm from the top (this usually takes 1–2 hours).

3. Mark the Solvent Front:

o When the solvent has nearly reached the top, remove the paper from the chamber.
 o Quickly mark the position of the solvent front with a pencil, as this will be used to calculate the retention factor (Rf) for each
base.

4. Visualizing the Nucleotide Bases

1. Dry the Chromatography Paper:

o Allow the paper to air dry in a well-ventilated area or use a hairdryer if time-sensitive.

2. Visualization:
o UV Light: If the nucleotide bases are fluorescent, place the dried paper under UV light to observe the spots.
o Staining with Ninhydrin (Alternative):
 Spray the chromatogram lightly with a ninhydrin solution (0.2% in acetone or ethanol).
 Heat the paper at 60–80°C for 5–10 minutes. Nucleotide bases should appear as colored spots due to their reaction with
ninhydrin.

3. Document the Results:

o Outline each visible spot with a pencil to document their positions.
o Photograph the chromatogram or record the positions if using a UV light.

5. Calculating Retention Factor (Rf) Values

1. Measure Distances:
o Measure the distance from the baseline to the center of each spot (distance traveled by the compound).
o Measure the distance from the baseline to the solvent front (distance traveled by the solvent).

2. Calculate Rf Value for Each Nucleotide Base:

Rf=Distance traveled by the compoundDistance traveled by the solvent frontRf = \frac{\text{Distance traveled by the compound}}{\
text{Distance traveled by the solvent front}}Rf=Distance traveled by the solvent frontDistance traveled by the compound

o Rf values are specific to the conditions of the experiment (solvent, temperature, and paper type) and can help identify the
compounds if compared with standard Rf values.

Troubleshooting Tips

 Spot Spreading: If spots are too large or spread out, apply smaller volumes and allow each layer to dry before reapplying.
 Poor Separation: Adjust the solvent ratio or change the paper type if the nucleotide bases are not well-separated.
 Faint Spots: Try increasing the concentration of nucleotide solutions or use a more sensitive visualization method.

Quick Reference Table

Step Reagents/Equipment Details

Solvent Preparation Butanol, acetic acid, water 4:1:5 ratio, mix thoroughly and add to chamber
Chromatography Paper Whatman No. 1 Mark baseline 2 cm from bottom, spot nucleotide bases
Developing Chromatogram Glass chamber, solvent Allow solvent to rise until 1–2 cm from top
Visualization UV light or ninhydrin Dry paper, visualize under UV or stain with ninhydrin
Rf Calculation Ruler Measure distance traveled by spots and solvent front