DNA Isolation: A Complete CSIR-NET Guide (Concepts, Steps & Exam Traps)

DNA isolation (also called DNA extraction) is one of the most fundamental techniques in molecular biology and a frequently tested topic in CSIR-NET, GATE, DBT-JRF, and university exams. Questions are asked not just on steps, but on the role of each reagent, principle, and experimental variations.

This blog explains DNA isolation step-by-step, with exam-oriented explanations and high-yield facts.

What is DNA Isolation?

DNA isolation is the process of separating DNA from cellular components such as:

• Cell wall
• Cell membrane
• Proteins
• RNA
• Lipids
• Polysaccharides

The final goal is to obtain pure, intact DNA suitable for downstream applications like:

• PCR
• Restriction digestion
• Cloning
• Sequencing
• Southern blotting

Principle of DNA Isolation (VERY IMPORTANT FOR CSIR-NET)

The principle is based on:

1. Cell lysis
2. Removal of proteins and contaminants
3. Precipitation of DNA

DNA is:

• Negatively charged
• Insoluble in alcohol (ethanol/isopropanol)
• Stable in slightly alkaline pH

General Steps of DNA Isolation

Cell Lysis (Breaking the Cell)

The first step is to break the cell wall and membrane to release DNA.

Sample Type	Method Used
Bacteria	Lysozyme + detergent
Plant cells	Grinding + CTAB
Animal cells	Detergents only

 Detergents used:

• SDS (Sodium dodecyl sulfate)
• CTAB (Cetyl trimethyl ammonium bromide)

CSIR-NET Tip:
CTAB is especially used for plant DNA isolation because it removes polysaccharides.

Removal of Proteins

After lysis, DNA is mixed with proteins (histones, enzymes).

 Common methods:

• Phenol–chloroform extraction
• Proteinase K digestion

Phenol–Chloroform Method

• Phenol → denatures proteins
• Chloroform → improves phase separation
• Centrifugation forms two layers:

Layer	Contains
Upper aqueous phase	DNA
Lower organic phase	Proteins

 Exam Trap:
DNA remains in aqueous phase, not organic phase.

 Removal of RNA

RNA contamination is removed using:

• RNase A

RNase is:

• Heat stable
• Does not require cofactors

DNA Precipitation

DNA is precipitated using alcohol.

Alcohol Used	Condition
Ethanol (cold)	2–2.5 volumes
Isopropanol	0.6–1 volume

 Salt required:

• Sodium acetate / NaCl

Why salt?
Neutralizes negative charge on DNA phosphate backbone.

CSIR-NET Favorite Question:
DNA precipitates because it is insoluble in alcohol.

Washing and Resuspension

• Wash DNA pellet with 70% ethanol
• Air dry
• Dissolve in:
• TE buffer (Tris-EDTA)
• Nuclease-free water

Role of EDTA:

• Chelates Mg²⁺
• Inhibits DNases

CTAB Method for Plant DNA (High-Yield Topic)

Plant cells contain:

• Polysaccharides
• Polyphenols

CTAB:

• Forms complexes with polysaccharides
• Prevents DNA degradation

β-mercaptoethanol is added to:

• Remove polyphenols
• Prevent oxidation

CSIR-NET Repeated Concept:
CTAB is preferred for plant genomic DNA isolation.

Quality and Quantity Check of DNA

UV Spectrophotometry

Ratio	Interpretation
A260/280 ≈ 1.8	Pure DNA
< 1.8	Protein contamination
> 2.0	RNA contamination

 DNA absorbs at 260 nm, proteins at 280 nm.

 Agarose Gel Electrophoresis

• Intact DNA → sharp high-molecular-weight band
• Smearing → degraded DNA

 Common CSIR-NET Exam Traps

✔ Phenol is corrosive
✔ EDTA inhibits DNase
✔ Isopropanol requires less volume than ethanol
✔ CTAB removes polysaccharides
✔ DNA is negatively charged
✔ RNase removes RNA, not DNA

One-Line CSIR-NET Memory Box

·         Lysozyme → bacterial cell wall

·         SDS → membrane lysis

·         Phenol → protein denaturation

·         RNase → RNA removal

·         Cold ethanol → DNA precipitation

·         EDTA → DNase inhibition

NA isolation is the process of extracting pure DNA from cells by removing membranes, proteins, and RNA.

Main Steps:

1.      Cell lysis – Detergents (SDS/CTAB) break cell membrane and wall.

2.      Protein removal – Phenol–chloroform or Proteinase K removes proteins.

3.      RNA removal – RNase digests RNA.

4.      DNA precipitation – Cold ethanol/isopropanol + salt precipitates DNA.

5.      Washing & resuspension – 70% ethanol wash; dissolve in TE buffer.

Key Exam Points:

·         CTAB → used in plant DNA isolation (removes polysaccharides).

·         EDTA → inhibits DNase by chelating Mg²⁺.

·         DNA precipitates in alcohol because it is insoluble.

·         Pure DNA A260/280 ≈ 1.8.

 CSIR-NET tip: DNA stays in the aqueous phase during phenol–chloroform extraction.

Conclusion

DNA isolation is not just a laboratory technique but a conceptual goldmine for CSIR-NET. Understanding:

• Why each reagent is used
• Which step removes what
• How purity is measured

will help you confidently solve assertion-reason, match-the-following, and numerical questions in the exam.

 

DNA Isolation – CSIR NET MCQ Quiz

1. The main purpose of EDTA in DNA isolation buffer is to:

Precipitate DNA
Denature proteins
Chelate divalent cations
Lyse cell membrane

2. Which detergent is commonly used to lyse cell membranes during DNA isolation?

SDS
Agarose
EDTA
Tris-HCl

3. Proteinase K is used in DNA isolation to:

Degrade RNA
Degrade proteins
Precipitate DNA
Chelate Mg²⁺ ions

4. Which reagent is used to remove RNA contamination?

RNase A
DNase I
SDS
Chloroform

5. DNA is precipitated commonly using:

Phenol
Ethanol or Isopropanol
Tris buffer
SDS

6. Phenol–chloroform extraction mainly removes:

DNA
RNA
Proteins and lipids
Salts

7. High salt concentration during DNA precipitation helps in:

DNA degradation
Protein solubilization
Neutralizing DNA charges
RNA digestion

8. A 260/280 ratio of ~1.8 indicates:

RNA contamination
Protein contamination
Pure DNA
Degraded DNA

9. Which enzyme degrades DNA if not inhibited during isolation?

RNase
Ligase
DNase
Polymerase

10. Tris-HCl buffer maintains:

Osmotic balance
pH stability
DNA precipitation
Protein digestion