“Cell lysis is one of the critical steps in DNA extraction. The process to break a cell wall or nuclear membrane using chemical, physical or enzymatic methods is known as cell lysis or lysis.”
Cell lysis is a must! I mean, without lysing a cell, we can’t isolate DNA. DNA isolation or DNA extraction is a process to extract or obtain DNA from cells. Usually, we use various chemicals or a combination of chemicals to do this. If you wish to learn more about DNA extraction you can read our previous articles (links were given in the article somewhere).
Coming to the point, different cell types have different extraction methods. For plant, animal, bacteria and blood DNA extraction, the process varies and so the composition of lysis buffer too. Also, the cell type differ as well, for example, plants have a thick pectin rich cell wall while bacterial has an only cell membrane.
So Practically, a more aggressive cell lysis method is needed in the case of plant DNA extraction. The present article explains the preparation of lysis buffer for different types of DNA extraction methods and its importance too.
In addition, we also give you a perfect recipe to prepare lysis buffer for animal, plant, bacterial and other DNA extraction protocols. Now let’s start the article.
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. The image below illustrates the general process of DNA extraction.
Preparation of lysis buffer for blood DNA extraction:
Two different combinations of solutions are used for lysis buffer preparation, especially for the blood samples. 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 ingredients in sterile D/W and set pH 7.6
Autoclave it and wait to come at room temperature.
add 0.5% SDS (0.250gm).
If you wish to learn the role of each chemical you should read this article: Phenol chloroform DNA extraction: Basics, preparation of chemicals and protocol.
Preparation of lysis buffer for plant DNA extraction:
CTAB (hexadecyltrimethylammonium bromide) is the major ingredient for plant DNA extraction. Pectin present in the plant cell wall makes it harder and so it is also difficult to digest it. Along with some other ingredients like Tris, EDTA, NaCl, PVP, Beta- mercaptoethanol and ascorbic acid, CTAB lysis the plant cell wall effectively.
In order to remove other debris, the combination of mechanical and enzymatic treatment is employed too along with the chemical lysis. Learn more on the present topic by reading this article: CTAB DNA extraction buffer for plant DNA extraction.
Here is the lysis buffer recipe for plant DNA extraction.
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:
The cell structure of bacteria is totally different from the plant, here a smooth cell membrane is present instead of a hard cell wall. And therefore lysing bacterial cell membranes is an easy task comparing to plant. The less chemical treatment gives good quality DNA here. Even, some bacterial lysis is performed only by heating.
Only the TE buffer is sufficient to lyse bacterial cell membrane. 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
Pro-tips: in the 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:
Plasmids are smaller viral DNA; circular in nature and present in a little amount. Though it is easy to extract plasmid DNA, I personally recommended using a ready-to-use plasmid DNA extraction kit. Or for manual processes, you can use TE buffer for lysis.
You can read our previous article on TE buffer to get more information on the present topic: Importance of Tris-EDTA (TE) buffer in DNA extraction.
Or
Read more on plasmid DNA: Plasmid DNA- Structure, Function, Isolation And Applications.
Preparation of 10X TE buffer for plasmid DNA:
100mM Tris HCl
10mM EDTA
“Breaking the code” of lysis buffer:
We have given a concentration of lysis buffer for different types of DNA extraction methods, but that isn’t enough! The objective to write this article is that “can you prepare your own lysis buffer for the DNA extraction method you use? ”
Let’s check it out;
First, understand the importance and role of every chemical used in DNA extraction as well as cell wall lysis. Now, let’s decode the puzzle of lysis buffer. Firstly, understand which chemicals are most common? Tris and EDTA, right!
Tris and EDTA are two main chemicals or a foundation 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 inactivates 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 in the cell membrane and nuclear membrane. Commercially available detergents can do that such as SDS and CTAB. Try one or two concentrations and use your own or I had given the concentration above, somewhere.
Third, use of 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. So that must also be included in the recipe.
These chemicals are the basic foundation. In addition, we can use beta-mercaptoethanol, ascorbic acid, TrioinX 100 and polyvinylpyrrolidone (PVP) too to increase the yield and purity of DNA.
Next time when you prepare any lysis buffer for any type of DNA extraction protocol don’t be scared just think about Tris, EDTA, SDS, CTAB, MgCl2 and other salts and use an appropriate concentration of it. It’s 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 using the 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 the phenol-chloroform DNA extraction method is explained in this article: phenol-chloroform DNA extraction method.
For plant and related DNA extraction use the proteinase K method. Enzymatic methods give more powerful results than any other method. Our protocol of the proteinase K method is here: proteinase K DNA extraction method.
Note: all the compositions are tried and checked in our lab by our experts and we are using them. You can directly use it.
Some of the interesting articles:
Conclusion:
As we discussed, cell wall structure and composition differ among organisms, so we need different combinations as well as different concentrations of chemicals. Tris, EDTA, detergents and salt are common among all.
Ready-to-use kits are the best alternative and give good results too, but I always prefer to use manual methods because you can create different combination and every time can learn something new. To sharpen your practical knowledge I advise using manual methods.
