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«Product Code: HTBM005 Number of experiments that can be performed: 10 Duration of Experiment Protocol: 3-4 hours Agarose Gel Electrophoresis: 1 hour ...»

.

Plant Genomic DNA Extraction Teaching Kit

(Solution Based)

Product Code: HTBM005

Number of experiments that can be performed: 10

Duration of Experiment

Protocol: 3-4 hours

Agarose Gel Electrophoresis: 1 hour

Storage Instructions:

The kit is stable for 6 months from the date of receipt

Store Control DNA at -20oC

Store 6X Gel Loading Buffer at 2-8oC

Other kit contents can be stored at room temperature (15-25oC)

1

Index

Sr. No. Contents Page No.

1 Aim 3 2 Introduction 3 3 Principle 3 4 Kit Contents 3 5 Materials Required But Not Provided 4 6 Storage 4 7 Important Instructions 4 8 Procedure 4 9 Agarose Gel Electrophoresis 6 10 Quantitation of DNA 6 11 Flowchart 7 12 Observation and Result 8 13 Interpretation 8 14 Troubleshooting Guide 9 2

Aim:

To extract and analyze genomic DNA from leaves by CTAB method.

Introduction:

DNA extraction from plant tissues, unlike DNA isolation from mammalian tissues, remains difficult due to the presence of a rigid cell wall surrounding the plant cells. DNA extraction from plant tissue can vary depending on the material used. Essentially any mechanical means of breaking down the cell wall and membranes to allow access to nuclear material, without its degradation is required. The CTAB (Cetyl trimethyl ammonium bromide) method can be used both for freeze dried leaves and for fresh leaves.

For this, usually an initial grinding stage is employed to break down cell wall material and allow access to DNA. Once the tissue has been sufficiently ground, it can then be resuspended in a suitable buffer, such as CTAB. In order to purify DNA, insoluble particulates are removed through centrifugation, while soluble proteins and other material are separated by mixing with chloroform:Octanol followed by centrifugation. DNA must then be precipitated from the aqueous phase and washed thoroughly to remove contaminating salts.

The purified DNA is then resuspended and stored in Tris EDTA buffer or sterile distilled water. This method has been shown to give intact genomic DNA from plant tissue. To check the quality of the extracted DNA, a sample is run on an agarose gel, stained with ethidium bromide, and visualized under UV light.

Principle:

CTAB (Cetyl trimethyl ammonium bromide) isa detergent used to break open plant cells and solubilize its contents. The extraction process involves breaking or digestion of cell wall in order to release the cellular constituents. This is followed by disruption of the cell membrane to release the DNA into the extraction buffer. The released DNA should be protected from endogenous nucleases. EDTA is often included in the extraction buffer to chelate magnesium ions, a necessary co-factor for nucleases. The initial DNA extracts often contain a large amount of RNA, proteins, polysaccharides, tannins and pigments which may interfere with the extracted DNA and difficult to separate. Most proteins are removed by denaturation and precipitation from the extract using chloroform and octanol. RNAs on the other hand are normally removed by treatment of the extract with RNase A. The DNA is precipitated and washed in organic solvents before re-dissolving in aqueous solution.

Kit Contents:

The kit can be used to perform plant DNA extraction using CTAB.

Table 1: Enlists the materials provided in this kit with their quantity and recommended storage

–  –  –

Glass wares: Conical flask, measuring cylinder Reagents: Distilled water, Ethidium bromide (10 mg/ml), β - Mercaptoethanol, Chloroform, Octanol, Ethanol Other requirements: Plant leaves, Electrophoresis apparatus, UV Transilluminator, Micropipettes, Vortex Mixer, Tips, Adhesive tape

Storage:

Plant Genomic DNA Extraction Teaching Kit (Solution Based) can be stored for up to 6 months without showing any reduction in performance. On receipt, store control DNA at -20oC and 6X Gel Loading Buffer at 2-8oC. Other kit contents can be stored at room temperature (15-25oC).

Important Instructions:

–  –  –

Procedure:

It is preferable to use young plant parts especially leaves, needles, since they contain more cells per weight and therefore result in higher yields. Also, young leaves and needles contain less polysaccharides and polyphenolics and are therefore easier to handle.

A) Sample Preparation:

Take young and tender leaves (for e.g. mint, spinach, tulsi, ginger etc) and wash them with distilled water.

Finely cut the leaf material. Midrib and petiole should be removed from the leaf material before grinding, as they are a major source of carbohydrate contamination.

Note: DNA is a large molecule that can be broken down by shear forces, care should be taken to mix the samples gently, never vortex the DNA.

–  –  –

B) DNA Extraction:

1. Grind 350 mg of freshly cut leaves in a mortar and pestle by adding 4 ml of prewarmed CTAB Extraction Buffer. Transfer the mixture to 15 ml centrifuge tube using a clean spatula.

Lysis 2.





Transfer the mixture to 15 ml centrifuge tube containing 5 ml prewarmed CTAB Extraction Buffer using a spatula. Mix gently by inversion.

Incubate the sample at 65oC for 60 minutes, with occasional inversion of the tube.

3.

Allow the sample to cool down by keeping the tubes at room temperature (15-25oC) for 5 minutes.

4.

Phase Separation 5.

Add 5 ml of Chloroform: Octanol (24:1) and mix by rocking the tube gently for 5 minutes.

Centrifuge the samples at 2,300 rpm for 2 minutes at room temperature (15-25oC).

6.

Transfer the top aqueous layer into a fresh 15 ml centrifuge tube and add 25 µl of RNase A. Mix the 7.

sample gently by inversion and incubate for 30 minutes at room temperature (15-25oC).

Precipitation of DNA 8.

Add 6 ml of isopropanol and mix the samples gently by inversion until a white fluffy DNA precipitate appears (it should appear within 1 minute after addition of isopropanol).

Centrifuge the samples at 2,300 rpm for 5 minutes at room temperature (15-25oC). Discard the 9.

supernatant.

10. Wash Add 8 ml of cold CTAB Wash Buffer to the sample and mix by pipetting. Incubate at room temperature (15-250C) for 20 minutes. Do not vortex as it may result in shearing of DNA.

11. Centrifuge the samples at 2,300 rpm for 5 minutes at room temperature (15-25oC). Discard the supernatant.

12. Add 8 ml of cold 70% ethanol to the tube containing the DNA and mix by pipetting. Centrifuge at 2,300 rpm for 5 minutes. Discard the supernatant.

–  –  –

14. Elution Add 1ml of Elution Buffer and resuspend the above pellet.

Storage of the DNA: For short-term storage (24-48 hours) of the DNA, 2-8oC is recommended. For longterm storage, -200C or lower temperature (-80oC) is recommended. Avoid repeated freezing and thawing of the sample which may cause denaturing of DNA. The Elution Buffer will help to stabilize the DNA at these temperatures.

Agarose Gel Electrophoresis:

Preparation of 1X TAE: To prepare 500 ml of 1X TAE buffer, add 10 ml of 50X TAE Buffer to 490 ml of sterile distilled water*. Mix well before use.

Preparation of agarose gel: To prepare 50 ml of 0.8 % agarose gel, add 0.4 g agarose to 50 ml of 1X TAE buffer in a glass beaker or flask. Heat the mixture on a microwave or hot plate or burner, swirling the glass beaker/flask occasionally, until agarose dissolves completely (Ensure that the lid of the flask is loose to avoid buildup of pressure). Allow the solution to cool to about 55-60oC. Add 0.5µl Ethidium bromide, mix well and pour the gel solution into the gel tray. Allow the gel to solidify for about 30 minutes at room temperature.

NOTE: Ethidium bromide is a powerful mutagen and is very toxic. Appropriate safety precautions should be taken by wearing latex gloves; however, use of nitrile gloves is recommended.

Loading of the DNA samples: To prepare sample for electrophoresis, add 2 µl of 6X gel loading buffer to 10 µl of DNA sample. Mix well by pipetting and load the sample onto the well. Load the Control DNA after extracting the DNA sample.

Electrophoresis: Connect the power cord to the electrophoretic power supply according to the conventions:

Red-Anode and Black- Cathode. Electrophorese at 100-120 volts and 90 mA until dye markers have migrated an appropriate distance, depending on the size of DNA to be visualized.

* Molecular biology grade water is recommended (Product code: ML024).

Quantitation of DNA:

Spectrophotometric analysis and agarose gel electrophoresis will reveal the concentration and the purity of the genomic DNA. Use Elution Buffer to dilute samples and to calibrate the spectrophotometer, measure the absorbance at 260 nm, 280 nm, and 320 nm using a quartz microcuvette. Absorbance readings at 260 nm should fall between 0.1 and 1.0. The 320 nm absorbance is used to correct background absorbance. An absorbance of 1.0 at 260 nm corresponds to approximately 50 µg/ml of DNA. The A260 - A320 /A280 -A320 ratio should be 1.6 –1.9. Purity is determined by calculating the ratio of absorbance at 260 nm to absorbance at 280 nm.

Concentration of DNA sample (µg/ml) = 50 x A260 x dilution factor

–  –  –

Perform Agarose Gel Electrophoresis. Visualize the DNA bands using UV Transilluminator and calculate the yield and purity using UV Spectrophotometer.

1 2 3

–  –  –

Lane 1: Control DNA Lane 2: Extracted plant DNA Lane 3: Plant DNA with RNA contamination Table 2: Absorbance of the extracted genomic DNA at 260 nm and 280 nm

–  –  –

Calculate the concentration of isolated DNA using following formula:

Concentration of DNA sample (µg/ml) = 50 x A260 x dilution factor

Interpretation:

The lanes 1 and 2 demonstrate that highly purified DNA has been obtained with no visible RNA contamination when electrophoresed on agarose gel. If RNA contamination is present, one would see a faint and smeary RNA band below the genomic DNA as shown in lane 3. RNA being of lower molecular weight than DNA runs faster than the genomic DNA. RNA contamination is observed when the RNase treatment has either been skipped or not been carried out properly.

–  –  –

Technical Assistance:

At HiMedia we pride ourselves on the quality and availability of our technical support. For any kind of Technical assistance, mail at mb@himedialabs.com

–  –  –

9



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