.
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) is a 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
Sr. No.
1
2
3
4
5
6
7
8
9
10
11
Product
Code
TKC008
DS0002
DS0003
DS0004
DS0040
DS0029
MB002
ML016
ML015
MB063
PW144
Materials Provided
Control DNA
CTAB Extraction Buffer
RNase A solution
CTAB Wash Buffer
Elution Buffer
CTAB Powder
Agarose
50X TAE
6X Gel Loading Buffer
Isopropanol
Centrifuge Tubes
3
Quantity
10 expts
0.11 ml
108 ml
0.3 ml
25 ml
12 ml
1.1 g
4.8 g
120 ml
0.05 ml
72 ml
33 Nos.
Storage
-20oC
RT
RT
RT
RT
RT
RT
RT
2-8oC
RT
RT
Materials Required But Not Provided:
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:
1.
Preheat the water bath or heating block to 65oC.
2.
Preparation of CTAB Extraction Buffer for 1 expt: Just before starting the experiment, add 90 µl of β mercaptoethanol and 90 mg of CTAB powder to 9 ml of CTAB Extraction Buffer in a 15 ml centrifuge
tube. Preheat the solution to 65oC.
3.
Preparation of CTAB Wash Buffer for 1 expt: To 2 ml of CTAB Wash Buffer add 6 ml of Ethanol (96100%)
4.
Chloroform: Octanol (24:1) preparations for 1 expt- To 4.8 ml of chloroform, add 200 µl of octanol.
Store in dark at room temperature.
5.
Preparation of 70% Ethanol for 1 expt: Add 5.6 ml of ethanol (96-100%) to 2.4 ml of distilled water.
6.
Prechill the CTAB Wash Buffer and 70% Ethanol prior to use.
7.
Clean the mortar and pestle with distilled water before use.
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.
4
Fig 1: Leaf showing the midrib and petiole
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.
2.
Lysis
Transfer the mixture to 15 ml centrifuge tube containing 5 ml prewarmed CTAB Extraction Buffer using
a spatula. Mix gently by inversion.
3.
Incubate the sample at 65oC for 60 minutes, with occasional inversion of the tube.
4.
Allow the sample to cool down by keeping the tubes at room temperature (15-25oC) for 5 minutes.
5.
Phase Separation
Add 5 ml of Chloroform: Octanol (24:1) and mix by rocking the tube gently for 5 minutes.
6.
Centrifuge the samples at 2,300 rpm for 2 minutes at room temperature (15-25oC).
7.
Transfer the top aqueous layer into a fresh 15 ml centrifuge tube and add 25 µl of RNase A. Mix the
sample gently by inversion and incubate for 30 minutes at room temperature (15-25oC).
8.
Precipitation of DNA
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).
9.
Centrifuge the samples at 2,300 rpm for 5 minutes at room temperature (15-25oC). Discard the
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.
5
13. Air dry the pellet for 10-15 minutes for the ethanol to evaporate.
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
6
Flowchart:
Weigh 350 mg of finely cut young and tender leaves
Grind the leaves in 4 ml of prewarmed CTAB Extraction buffer
Lysis
Transfer the ground contents into 15 ml centrifuge tube containing 5 ml of
prewarmed CTAB Extraction buffer
Incubate for 60 minutes at 65oC.
Stand the tubes for 5 minutes at room temperature
Phase Separation
Add 5 ml of Chloroform:Octanol (24:1), mix gently for 5 minutes
Centrifuge for 2 minutes at 2,300 rpm
Transfer top aqueous layer to new tube, add 25 µl of RNase A.
Mix by gentle inversion
Incubate for 30 minutes at room temperature
Precipitation of DNA
Add 6 ml of isopropanol.
Mix by gentle inversion till white DNA precipitate appears
Centrifuge for 5 minutes at 2,300 rpm and discard the supernatant
Wash using CTAB Wash Buffer
Add 8 ml of cold CTAB Wash Buffer, mix by gentle pipetting
Incubate for 20 minutes at room temperature, do not vortex
Centrifuge for 5 minutes at 2,300 rpm (discard supernatant)
Wash using 70% Ethanol
Add 8 ml of 70% of ethanol, mix by pipetting.
Centrifuge for 5 minutes at 2,300 rpm (discard supernatant)
DNA Elution
Air dry the pellet for 10-15 minutes
Resuspend above pellet in 1 ml of Elution Buffer
Pure DNA
7
Observation and Result:
Perform Agarose Gel Electrophoresis. Visualize the DNA bands using UV Transilluminator and calculate the
yield and purity using UV Spectrophotometer.
1
2
3
Plant Genomic DNA
RNA
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
Sample
1
2
3
Dilution Factor
A260
A280
A260/A280
Concentration (µg/ml)
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.
8
An absorbance of 1.0 at 260 nm corresponds to approximately 50 µg/ml of DNA. If the A260/A280 ratio is 1.61.9, then the isolated DNA sample is considered to be pure. If higher A260/A280 ratio is observed it indicates the
possibility of RNA contamination.
Troubleshooting Guide:
Sr.
No.
Probable Cause
Solution
Grinding of the
midrib along with
the leaf material
Remove the midrib from the leaf before grinding,
mainly in case of maize and lettuce, as these plant
species have pronounced midribs. Removal of the
midrib is not important in case of very young leaves
or in small grain cereals
2
DNA appears
degraded (as a smear
running down the gel)
DNA appears
fragmented or
broken
DNA being a large molecule can be broken by shear
forces if treated vigorously. Therefore mix the
samples gently, never vortex the DNA. To minimize
shearing always use a wide bore pipette tip for
mixing
3
Difficult to dissolve
DNA in Elution Buffer
This is due to
over-drying of
DNA pellet
The DNA should not be allowed to over-dry at any
stage during the preparation as it hinders the
resuspension and solubilization in Elution Buffer
1
Problem
Carbohydrate
contamination in the
sample
Technical Assistance:
At HiMedia we pride ourselves on the quality and availability of our technical support. For any kind of
Technical assistance, mail at [email protected]
PIHTBM005_O/1013
HTBM005-03
9