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CTAB Extraction

This document provides a method for extracting genomic DNA from plant tissue using a CTAB buffer and quantifying the extracted DNA. Key steps include homogenizing tissue in CTAB buffer, incubating at 65°C, precipitating the DNA with isopropanol, and resuspending in TE buffer. A NanoDrop spectrophotometer is used to quantify DNA concentration based on absorbance at 260nm, and agarose gel electrophoresis confirms DNA quality and integrity.

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Janikaa Singaravel Murugan
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67 views3 pages

CTAB Extraction

This document provides a method for extracting genomic DNA from plant tissue using a CTAB buffer and quantifying the extracted DNA. Key steps include homogenizing tissue in CTAB buffer, incubating at 65°C, precipitating the DNA with isopropanol, and resuspending in TE buffer. A NanoDrop spectrophotometer is used to quantify DNA concentration based on absorbance at 260nm, and agarose gel electrophoresis confirms DNA quality and integrity.

Uploaded by

Janikaa Singaravel Murugan
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as DOCX, PDF, TXT or read online on Scribd
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Plant genomic DNA extraction by CTAB method and Quantification

Aim:

To extract the genomic DNA from the given sample

Principle:

Isolation of plant genomic DNA is a basic requirement for genome characterization, mapping
and isolation of genes for genetic engineering. Irrespective of methods, DNA extraction involves
three major steps viz, disruption of the cell wall; de-proteinization of an aqueous solution
containing the DNA, precipitation and purification of the DNA. If the DNA is to be extracted, a
pH of 8.0 or higher must be used. The reason is that at lower pH, DNA is selectively retained in
the organic phase leaving the RNA in the aqueous phase. De-proteinization is done using the
organic compounds such as phenol, chloroform or isopropanol. The use of chloroform improves
the efficiency of DNA extraction and isoamyl alcohol is added to chloroform to prevent foaming.

Requirements

1.Fresh leaf sample


2. Liquid N2
3. Mortar and pestle
4.CTAB buffer
 Tris-Hcl, pH 8.0-100mM
 EDTA-20mM
 NaCl-1.4M
 2%CTAB (W/V)-0.5%
 β- mercaptoethanol-0.1%
5.chloroform: iso-amyl alcohol (24:1)
6. ice cold isopropanol
7.70% ethanol
8.1X TE Buffer
9.Agarose
Procedure

1. Homogenize 200 mg of plant tissue to a fine paste with 500 μl of CTAB buffer.
2. Transfer CTAB/plant extract mixture to a microfuge tube.
3. Incubate the CTAB/plant extract mixture for about 45 min at 65˚C in a re-circulating water
bath.
4. After incubation, spin the CTAB/plant extract mixture at 12000 rpm for 5 min to spin down
cell debris. Transfer the supernatant to clean microfuge tubes.
5. Add 2-5ul of RNase A to the transferred supernatant.
6. To each tube add an equal volume of Chloroform: Iso Amyl Alcohol (24:1) and mix the
solution by inversion. After mixing, spin the tubes at 12000 rpm for 1 min.
7. Transfer the upper aqueous phase only (which contains the DNA) to a clean microfuge tube.
8. To each tube add 500 μl of ice cold isopropanol.
9. Invert the tubes slowly several times to precipitate the DNA. Generally, the DNA can be seen
to precipitate out of solution. Alternatively, the tubes can be placed for 2 hours at -20˚C to
precipitate the DNA.
10. Centrifuge at 5000 rpm for 10 minutes to pellet the precipitate.
11. To wash the DNA, transfer the precipitate into a microfuge tube containing 200 μl of ice-cold
70 % ethanol and slowly invert the tube. The precipitate can be done by spinning the tube at
12000 rpm for a minute to form a pellet.
12. Discard 70% ethanol from tubes, invert the tube containing DNA pellet on tissue paper, and
allow tubes containing pellet to air dry at room temperature (approximately 15 min).
13. Resuspend the DNA in sterile TE buffer. Then the DNA is stored at -20˚C for further
experiments.
14. Agarose gel electrophoresis of the DNA will show the integrity of the DNA, while
spectrophotometry will give an indication of the concentration and purity.

Quantification of genomic DNA using NanoDrop/Spectrophotometer

1. Before measuring samples, ‘blank’ the spectrophotometer using the solution the DNA is
resuspended in, but with no DNA added. 'Blanking' measures the background inherent to
the machine and your solvent.
2. To measure the samples, place 1-2µL sample (DNA) onto the pedestal.
3. Close the lid and click measure, record the concentration and purity.

Nucleic acids (DNA and RNA) absorb maximally at 260 nm. Proteins on the other hand absorbs
at 280nm and organic compounds and chaotropic salts maximally absorb at 230nm. The
A260/A280 ratio is used as an indicator of DNA purity. Ideally, this number should be between
1.8 and 2.0. The A260/A230 ratio is best if greater than 1.5.
Then, using the A260 reading, you can calculate the DNA concentration. Generally, A260 of 1.0
is equivalent to 50 ug/ml pure dsDNA. Use the following formula to estimate your DNA:

Concentration (µg/ml) = A260 reading x dilution factor x 50 µg/ml

DNA quality confirmation

1. Prepare a 0.8 % solution of agarose by melting 0.8 g of agarose in 100 mL of 1X TAE buffer
in a microwave oven for approximately 2 min. Allow to cool for a couple of minutes then add
2.5 μl of ethidium bromide, stir to mix.
2. Cast a gel using a supplied tray and comb. Allow the gel to set for a minimum of 20 min at
room temperature on a flat surface. Run the gel for 30 min at 100 V
3. Expose the gel to UV light and photograph (Gel documentation).
4. Confirm DNA quality, presence of a highly resolved high molecular weight band indicates
good quality DNA, presence of a smeared band indicates DNA degradation.

Result and Discussion:

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