Plant DNA Extraction – CTAB DNA Extraction Protocol

Deoxyribonucleic acid  is thread like structure made up of nucleotide having genetic information. This is found in every organism. DNA is a heredity material. The information is stored in the form of codes. These codes made up of four type of bases. The important thing is that DNA can replicate and can make copies. And its sequence can be find out using tools like Expasy Translate Tool.

What is DNA?

DNA strands for Deoxyribonucleic acid

How DNA is formed?

DNA is formed by double strand of polynucleotide. Which consist of 4 different type of nucleotide. Hydrogen bond is present in between base protein of nucleotide.

Guanine, cytosine, adenine, thiamin   are 4 different types of bases are present in DNA which are linked together through hydrogen bond.

Adenine and thiamin has double bond and Guanine and cytosine has triple bond. These two strands raped to each other one strand of Deoxyribonucleic acid linked through hydrogen bonding with other strand.

Why Deoxyribonucleic acid is important?

This is a nucleic acid which has genetic information and this genetic information is used for development and functioning of all living organism DNA storage all type of genetic information. This genetic information is carrying through gene.

Deoxyribonucleic acid is found in living organisms in any part of body it is a thread like structure.

It is so important in many cases like DNA finger printing, crime scene, parents indication with child, siblings identification etc.

Chemical structure of DNA

Use of DNA extraction Extraction is used to diagnose many medical conditions and also used for genetic engineering of both plant and animals.

Where DNA is located?

In organisms called eukaryotes, DNA is found inside a special area of the cell called the nucleus.cell studies in cell Biology Branch of Biology. Because the cell is very small, and because organisms have many DNA molecules per cell, each Deoxyribonucleic acid molecule must be tightly packaged. This packaged form of the DNA is called a chromosome.

During DNA replication, It unwinds so it can be copied. At other times in the cell cycle, It also unwinds so that its instructions can be used to make proteins and for other biological processes. But during cell division, It is in its compact chromosome form to enable transfer to new cells.

Researchers refer to DNA found in the cell’s nucleus as nuclear DNA. An organism’s complete set of nuclear DNA is called its genome.

Besides the Deoxyribonucleic acid located in the nucleus, humans and other complex organisms also have a small amount of Deoxyribonucleic acid in cell structures known as mitochondria. Mitochondria generate the energy the cell needs to function properly.

In sexual reproduction, organisms inherit half of their nuclear DNA from the male parent and half from the female parent. However, organisms inherit all of their mitochondrial DNA from the female parent. This occurs because only egg cells, and not sperm cells, keep their mitochondria during fertilization. [1]

Backbone of DNA made of

The “backbone” of Deoxyribonucleic acid is formed by sugar-phosphate-sugar chain and nitrogenous bases are faced inside. Within a polynucleotide chain, the two nucleotides are linked by phosphodiester bond. In nucleotide, phosphoric acid and pentose sugar( deoxyribose) is bonded with phosphoester bond. And pentose sugar and nitrogenous bases are bonded with N-Glycosidic bond. The two strands of DNA are held together by Hydrogen bond which are formed between nitrogenous bases of opposing strand. [2]

CTAB Deoxyribonucleic acid [DNA] extraction protocol from plant cell.

HOW to make solution which is used in DNA extraction

  • CTAB buffer
  • 100 ml 1M Tris HCl pH 8.0
  • 280ml 5M Nacl
  • 40ml of 0.5 M EDTA
  • 20g of CTAB (cetyltrimethyl ammonium bromide)

Bring total vol to 1L with ddH2O

  • 5 M Nacl
  • 292.2g of Nacl
  • 700ml of H2O

Add Nacl by parts and bring 1L solution

  • 75 M ammonium acetate
  • 57.81 g of ammonium acetate
  • ~ 50 ml of H2O

Bring to 100 ml total volume

  • 0.5 EDTA
  • 186012 g EDTA
  • Add about 700 ml H2O
  • 16- 18 g of NaOH pellets
  • Adjust pH to 8.0 by with a few more pellets; EDTA won’t dissolve until the pH is near 8.0

Bring total vol to 1 Lwith ddH2O

  • 1 M tris HCl pH 8.0
  • 121.1 g tris Hcl
  • Dissolve in about 700 of H2O
  • Bring pH down to 8.0 by adding concentrated GCl

Bring total volume 1L with dd H2O

  • TE buffer
  • 10ml 1M TRis HCl pH8.0
  • 2ml 0.5 M EDTA

Bring total volume to 1 L with ddH2O

CTAB DNA Extraction Principle

isolation of DNA from Plant cell

  • Prepare CTAB buffer prior to starting extraction, add polyvinylpyrrolidone and b-mecaptoethanol. Ince these have been added the shelf life of the buffer is only 2-3 days.

Preparation of CTAB buffer for DNA excretion

CRAB buffer
PVP
B-mercaptoethanol
0.5ml 0.02g 2.5 ul
5ml 0.2g 25ul
20ml 0.8g 100 ul
  • Grinding 50- 60 mg of plant tissue in liquid nitrogen , keeping tissue frozen the entire time. Transfer ground plat tissue in eppendrop and label it with permanent.
  • Add 500 ul CTAB buffer and mix well. Make sure the leaf tissue is in solution and not in a clump at bottom of the tube. Incubate at 55 degree C for at least one hour, mixing once after 30 minutes. They can stay in water bath for a few hours if necessary.
  • Optional stepAfter incubating for 1 hour, cool the mixture to room temperature and add 1.5 ul RNase A. Incubate at 37 degree for 15 minutes. This step should be included if you want clean, RNA free extractions.

The following steps are best done in bathes of 10-20, depending on how quickly you can work.

  •  Remove samples from water/ dry bath in FUME HOOD, add 500 ul of chloroform and mix by gently shaking tubes. Change gloves immediately if you spill chloroform on them. Be careful not to drip chloroform onto the tubes, it will remove the labeling.
  •  Centrifuge for 7 minutes at 1000 rpm.
  • Transfer the aqueous phase (top layer) into the new labeled tube. Be careful to avoid transferring any chloroform. You can tell if you get chloroform because it will be bright green. Chloroform waste should be disposed of in la proper way.
  •  Estimate the volume of the aqueous phase. Add 0.08 volumes cold 7.5 M ammonium acetate.
  • Add 0.54 volume of cold isopropanol mix by  inverting tubes 20-30 times.
Aqueous phase ul
Ammonium acetate
Isopropanol
350 28 204.12
360 28.8 209.952
370 29.6 215.784
380 30.4 221.616
390 31.2 227.448
400 32 233.448
410 32.8 239.112
420 33.6 244.944
  •  Incubate on ice for 30-40minutes
  • Centrifuge for 3 minutes at 13000 rpm
  • Discard supernatant into waste jar. Be careful not to dislodge pellet.
  • Add 700 ul 70% EtoH , invert tubes 5-10 times.
  • Centrifuge for 1 minute at 13000rpm.
  • Discard supernatant; be careful not to dislodge pellet.
  • Add 700 uL 95% EtoH , invert tubes 5-10 times.
  • Centrifuge for 1 minute at 13000 rpm.
  • Discard supernatant; be careful not to dislodge pellet.

Invert tubes on clean tissue paper and allow drying for 10-15 minutes upside down, or until pellet looks drying. If pellet dried too long upside down , it will fall out.

Hydrate pellet with 50ul TE. Allow to resuspend overnight at room temperature store the DNA in the refrigerator the next day.

If you won’t be in the next dna, make sure you ask someone else to do it.

References:

[1] https://www.genome.gov/25520880/deoxyribonucleic-acid-dna-fact-sheet/

[2] https://www.quora.com/What-is-the-backbone-of-DNA-made-of

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