How to prepare lysis buffer for different types of DNA extraction methods?

How to prepare lysis buffer for different types of DNA extraction methods?


A crucial step in genomic DNA extraction in any DNA extraction protocol is to lyse the cell. Breaking the cell wall or nuclear membrane with the help of different types of chemical, physical and enzymatic method is called as lysis or cell lysis.

 

In this article, we are discussing different types of cell lysis buffer used in different methods of DNA extraction. From organism to organism the DNA extraction methods vary which means the composition of lysis buffer also varies.

 

Composition and types of cell wall differ from organism to organism as well. For example, the cell wall of the plant cell is very thick due to the presence of pectin, contrary, the bacteria lack a cell wall, only have a soft cell membrane.   

 

Therefore from organism to organism, the composition of the lysis buffer for DNA extraction differs.

 

different types of DNA extraction methods
The image represents the general outline of how DNA extracted from the cell.

In this article, we are going to discuss different types of lysis buffer for DNA extraction of different types of an organism such as animal, plant, bacteria and virus.

 

Importance of lysis buffer for DNA extraction:

  • It lyses the nuclear membrane as well as a cell membrane.
  • It maintains the pH during the DNA extraction.  
  • Lysis buffer maintains the integrity of the DNA (protect DNA from lysis)
  • It separates DNA from other cell debris.
  • It protects DNA from acidic degradation.

General chemicals used in lysis buffer are Tris, EDTA, SDS, CTAB, Triton X100, MgCl2, KCl, NaCl and other detergents.

different types of DNA extraction methods
The general flowchart of the DNA extraction procedure.

 

Preparation of lysis buffer for blood DNA extraction:

 

The lysis buffer for extracting DNA from the blood is divided into two parts: solution I and solution II. The major components of the lysis buffer for blood DNA extraction are Tris, EDTA, MgCl2, KCl, NaCl and SDS.

 

Solution – I    (For 250ml)


10mM Tris (0.061 gm)
10mM KCl (0.186 gm)
10mM MgCl2 (0.238 gm)
Make-up final volume with D/W and set pH 7.6

 

Solution- II (50ml)

 

10mM Tris (0.061gm)
10mM KCl (0.037gm)
10mM MgCl2 (0.048gm)
0.5M NaCl(1.461gm)
2mM EDTA(0.037gm)
Mix all components in sterile D/W and set pH 7.6
Autoclave it and wait to come at room temperature.
0.5% SDS (0.250gm).

 

we had explained the role of each chemical into the article: Phenol chloroform DNA extraction: Basics, preparation of chemicals and protocol.

Read further on agarose gel electrophoresis:

  1. Agarose gel electrophoresis
  2. Agarose gel electrophoresis buffer
  3. DNA gel loading dye
  4. Role of EtBr in agarose gel electrophoresis

Preparation of lysis buffer for plant DNA extraction:

 

CTAB (hexadecyltrimethylammonium bromide) is the major ingredient for the DNA extraction of the plant cell. Due to the presence of the hardest pectin, it is not possible to break the cell wall of the plant cell. CATB facilitate to do so with some other chemicals such as Tris, EDTA, NaCl, PVP, Beta- mercaptoethanol and ascorbic acid.

 

Furthermore, mechanical lysis and enzymatic treatment are also required in plant DNA extraction. We will discuss plant DNA extraction protocol in some other article.

 

Here, the lysis buffer is divided into two parts,

Solution A (200ml)

2% CTAB (4.0 g)
100 mM Tris (pH 8.0) (20 ml)
20 mM EDTA (2 ml)
1.4 M NaCl (16.4 g)
4% polyvinylpyrrolidone (PVP) (8.0 g)
0.1% ascorbic acid (0.2 g)
10 mM β-mercaptoethanol (140 µL)

Solution B (200ml)

100 mM Tris-HCl (pH 8.0) (20 mL)
50 mM EDTA (10 mL)
100 mM NaCl (0.12 g)
10 mM β-mercaptoethanol (140 µL)

 

Preparation of lysis buffer for bacterial DNA extraction:

 

Instead of a cell wall, the bacterial cell contains a cell membrane. briefly, the cell membrane of the bacterial cell is much smoother than any other cell membrane/ cell wall. Therefore, it is easy to break the cell membrane of the bacterial cell. Fewer chemicals are required to achieve DNA extraction for bacterial DNA lysis buffer.

 

Only the TE buffer is sufficient to do so. However, a pinch of SDS along with the TE buffer increases the yield. The composition of the lysis buffer for the bacterial cell is as followed,  

Lysis buffer (100 ml),

10% SDS (10 ml)
90 ml TE buffer

How to prepare lysis buffer for different types of DNA extraction methods?
Image credit: www.mindsofmalady.com

Tips: in case of bacteria such as Tuberculosis, only heating can break the cell membrane of the bacteria. Heat the sample, centrifuge it, collect the pellets and directly use it in the PCR.

 

Preparation of lysis buffer for plasmid DNA extraction:

 

The virus contains very less DNA as compared with other organisms. So it is very crucial to isolate DNA preciously from the virus. For more sensitive experiments I recommended to use ready to use plasmid DNA extraction kit. Still, you can use the TE buffer directly as a lysis buffer for the plasmid DNA.

 

We had covered an article on the preparation and the function of TE buffer in DNA extraction please read the article here: importance of Tris-EDTA (TE) buffer in DNA extraction.

Preparation of 10X TE buffer,

100mM Tris HCl
10mM EDTA

 

Also, we had written an article on how to calculate the concentration of the chemicals for any biological reaction. Read the article here: Function of dNTPs in PCR reaction.

 

Read further on PCR,

  1. The Function of dNTPs in PCR reaction
  2. Role of DMSO in PCR: DMSO a PCR enhancer
  3. Function of taq DNA polymerase in PCR
  4. PCR primer design guidelines
  5. Role of MgCl2 in PCR reaction

“Breaking the code” of lysis buffer for DNA extraction:

 

we had given concentration for all the types of DNA samples, in this article. But that is not the purpose of writing this article. The main objective of the article is that “can you prepare your own lysis buffer for the DNA extraction?”

How to prepare lysis buffer for different types of DNA extraction methods?

 

To fulfil this aim, we have to understand the importance of each chemical, why each chemical is used into the lysis buffer and why not others.

 

Now, let’s decode the puzzle of lysis buffer. Firstly, understand which chemicals are most common? Tris and EDTA, obviously.  

 

Tris and EDTA are two major foundations in any DNA extraction procedure. We had covered an interesting article on Tris- EDTA buffer. Please first read the article here: importance of Tris-EDTA (TE) buffer in DNA extraction.

 

In short, Tris maintains the constant pH and EDTA chelates the metal ion and inactivate DNase and RNase.

 

So when you prepare any lysis buffer first include tris and EDTA. The concentration is given above.

 

Secondly, we have to degrade the proteins present into the cell membrane and nuclear membrane. Commercially available detergents can do that such as SDS and CTAB. Try one or two concentration and use your own or I had given the concentration above.

 

Third, some salts. MgCl2 protects DNA by blocking the negative charge of the lipoproteins and salts such as NaCl and KCl neutralize the charges on DNA and helps DNA to pull out of the cell. We had explained the role of each chemical in the article: different types of DNA extraction methods.

 

These chemicals are the basic foundation. In addition, we can use beta-mercaptoethanol, ascorbic acid, TrioinX 100 and polyvinylpyrrolidone (PVP) etc.

 

Next time when you prepare any lysis buffer for any types of DNA extraction protocol don’t be sacred just think about Tris, EDTA, SDS, CTAB, MgCl2 and other salts and use appropriate concentration of it. It is as easy as making a sandwich.

 

Now the next part of this article is how to use it.  You can choose any of the DNA extraction methods but I recommended to use phenol-chloroform DNA extraction method or proteinase K DNA extraction method.

 

For blood and any other animal body tissue use lysis buffer with phenol-chloroform DNA extraction method. Our protocol for phenol-chloroform DNA extraction method is here please follow it: phenol-chloroform DNA extraction method.

 

For plant and any other DNA extraction use the proteinase K method. Enzymatic methods give more powerful results than any other method. Our protocol of proteinase K method is here:  proteinase K DNA extraction method.

 

I always prefer to use manual methods because that will boost the practical knowledge otherwise you can use ready to use kits. Kits are more reliable than manual method but you can not gain anything out of it. So learn manual methods and be an expert.

 

Note: all the composition are tried and checked in our lab by our experts and we are using it. You can directly use it.  

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Conclusion:

In short, the concentration of each chemical might be different for different types of DNA extraction methods but Tris, EDTA, detergents and salts are the kings of any lysis buffer.