The DNA ladder is a standard sized fragment of DNA used to determine the molecular weight of the PCR amplicons. The commercially available DNA ladders come under 50bp, 100bp, 1000bp and 3000bp form.
Broadly, it is categorised into the standard molecular weight size marker. DNA ladder, RNA ladder and protein ladder are used for the sizing of DNA, RNA and protein respectively.
Those who are working in the field of genetics may already know about it. But here in the present article, we are talking about not only DNA ladder but how you can prepare it in your own research lab.
We are going to explain the method which you can use to make your own DNA ladder.
The content of the article is,
- What is a DNA ladder?
- Effect of the gelling conditions on DNA ladder
- How to make a DNA ladder? a DIY guide
Read our amazing article on DNA (a pictorial guide): DNA story: The structure and function of DNA
What is a DNA ladder?
The DNA ladder is a solution of different molecular weight fragments having double-stranded DNA used to determine the molecular weight or fragment size of the unknown PCR fragments.
Now, let’s understand the importance of the DNA ladder using an example. See the image given below.
The image is the results of agarose gel electrophoresis having different fragments of PCR amplicons. When we have not used a DNA ladder, is it conclusive?
No. it is not, because it is like sailing a sea without the compass.
But when we use a DNA ladder, we can conclude something, Right!! See the below image,
We can compare our PCR amplicons with the standard size DNA fragments having a known molecular weight.
An ideal DNA ladder must have several characteristics which are required to complete the electrophoresis experiments.
- Different fragments of the DNA ladder must be separable from each other.
- The concentration of each fragment must be sufficient enough to visualise on the gel.
- If the DNA ladder contains the DNA gel loading dye in it, the component of gel loading dye must not affect the specificity of the DNA ladder.
- It must be stable enough to use for a longer period of time.
- The ladder must be highly purified (prefer chromatography-based purified DNA ladder). Don’t use it, if it contains some unnecessary or unknown fragments (in that case contact the manufacturer).
Effect of the gelling conditions on DNA ladder:
Our standard molecular weight DNA marker comprises DNA fragments of different sizes. It is as like the DNA fragments of our PCR product. Therefore it can be affected by the gelling conditions as well.
Proper migration and separation of each fragment of a DNA ladder are two important factors must be achieved for getting good results.
The concentration of a buffer, Voltage, concentration of a gel and components of DNA gel loading dye are some of the conditions affect the migration of the DNA ladder.
The buffer or electrophoresis buffer is of two types, TAE buffer and TBE buffer. The concentration of each component used in it is given in our previous article: Agarose gel electrophoresis buffer.
Ideally, the 1X concentration of gel electrophoresis buffer is sufficient, If the concentration of buffer increases, it hinders the migration and separation of the DNA ladder.
Sharp and smooth DNA bands will not appear.
Voltage is another factor that is as crucial as the buffer.
Ideally, 5 to 10V/cm gel is recommended by the experts, however, if the voltage increases DNA fragments are trying to migrate fast resulting in the shearing DNA bands.
Therefore run a gel on an appropriate voltage condition. Ideally, 50 to 80V for 2% gel is highly recommended.
The concentration of a gel:
Yet, another important factor for DNA ladder to run properly is the concentration of the gel. If the concentration of gel is too high, the DNA fragments can not migrate properly.
For instance, if you are running a 2% agarose gel don’t use 3000bp (3kb) DNA ladder because 3kb fragment can not migrate properly into it. Even, the same DNA ladder cannot work for the agarose gel electrophoresis of restriction digestion product.
Contrary, the 10bp or 50bp fragment of DNA ladder cannot be properly separated on the 2% gel. Even, after the completion of a run, the fragment might not be visible.
The 10bp or 50bp DNA ladder is only useful for 3% gel (restriction digestion) and PAGE.
PAGE can separate smaller DNA fragments sharply.
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100bp DNA ladder:
In the 100bp DNA ladder, the smallest fragment is of 100bp.
The other fragment varies from the manufacturer to manufacturer. See the image given below,
Ideally, the 100bp DNA ladder contains 10 fragments of 100bp, 200bp, 300bp, 400bp,500bp, 600bp, 700bp, 800bp, 900bp and 1000bp.
However, some manufacturer provides 100 to 1500bp.
Ideally, the concentration of each DNA fragment is ~25ng except 500bp and 1000bp.
The concentration of the 500bp and the 1000bp fragment is 100ng, therefore, it can look sharper and more concentrated than other fragments.
The 100bp DNA ladder is one of the commonly used standard molecular weight markers during the PCR.
2% agarose gel concentration is ideal for it, although, 2.5% concentration of gel gives more sharpen DNA bands (our own observations).
1000bp (1kb) DNA ladder:
The 1000bp DNA ladder is not so often used in the routine PCR because the PCR products normally are of 100 to 900bp or maximum 1500bp.
The 1000bp ladder contains 10 fragments ranging from 1000 to 10kb.
On the 2% agarose gel it is hard to separate fragments of 5000bp or 10,000bp. However, it is used in the long-range PCR in which the amplicon size is larger than 5000bp.
For that, we have to set up a special agarose gel electrophoresis experiment. For the long-range PCR product, instead of 2% gel, 2.5 or 2.8% gel is advised because it separates the larger fragments very sharply.
Furthermore, the gel must run at a lower voltage, it also helps in proper separation.
How to make a DNA ladder? a DIY guide
3 different techniques are most popular to do so.
- Ligation method
- Restriction digestion
- PCR amplification
The ligation-based method is one of the traditional methods not used nowadays.
In the ligation-based DNA ladder development, different fragments of 100bp are covalently joined by the phosphodiester bonds that generated different fragments of 200bp to 1000bp.
See the image,
In the restriction digestion method, the know restriction endonuclease is used to digest different fragments of DNA that generate different sized fragments of DNA.
The digested fragments are collected, purified and can be used as a DNA ladder.
However, both the methods are outdated, costlier and time-consuming. In addition to this, the amount of fragment generated from both methods is very low.
Therefore, the need for the new method has arisen which may be cheap, rapid and most importantly generates a large amount of specific DNA fragments for the construction of DNA ladder.
We know that only PCR can generate millions of fragments in a short time.
PCR is used to generate different types of DNA fragments for the construction of a DNA ladder.
In the very first step, we have to select the plasmid. Use bacteria phage plasmid.
Now digest the plasmid with the appropriate restriction endonuclease so that the circular DNA breaks open.
Here we have selected lambda phage DNA sequence between the sequence 6631 to 7630 and designed primers according to it.
We have designed a set of primers that can amplify different fragments of DNA in a multiplex reaction.
The complete set of primers are given into the table below,
For achieving amplification for all the fragments, we need an advance PCR reaction preparation set up.
For instance, the R1 primer is used in all the amplification, therefore we have to add it 10 times more than other primers.
The reaction preparation for the present article is given into the table below,
|PCR buffer (50mM KCl, 2 mM MgCl2, 10mM Tris-HCl)||1X|
|F (forward primer||10pM each|
|R1 (reverse primer)||75-80pM|
|Taq DNA polymerase||5U|
|Nuclease-free water||As per requirement|
You can also read our PCR reaction preparation guide to learn how to prepare an effective PCR reaction: PCR reaction: Ten secrets that nobody tells you
The present method is a combination of three different PCR methods: Hot start PCR, multiplex PCR and touch down PCR.
Amplification of all the fragments are carried out in a single reaction (multiplex), Temperature gradually decreases to increase the amplification power of PCR (touch down) and the Taq DNA polymerase used in the present experiment is added only when the reaction is started (hot start).
Interestingly we had covered an in-depth article on all three PCR types. You can read it here before going further:
|PCR Steps||Initial Denaturation||Denaturation||Annealing||Extension||Final extension|
|Temperature||90 ̊C-95 ̊C||90 ̊C-95 ̊C||56 ̊C-44 ̊C||72 ̊C||72 ̊C|
|Time||5min||50 sec||50sec||2min||7 min|
Note: The method is a touchdown PCR, therefore, decrease the temperature after 2 PCR cycles until the temperature reaches 44 ̊C.
After the completion of the PCR reaction, the products are loaded on the 2% agarose gel.
Want to learn how to prepare an agarose gel? read this article: Agarose gel electrophoresis
Run the gel on 80V until the DNA reaches up to the 75% distance of the gel.
10 different DNA bands are obtained from the PCR reaction and are compared with the ready to use DNA ladder.
Now, our DNA ladder is ready to use, but before that, we have to purify the fragments.
For that precipitate the fragments using the alcohol and purify it with the ready to use DNA purification kit.
You can also use the phenol-chloroform method for DNA purification (Here I prefer to use a kit based method over the PCI method).
Again dissolve the fragments in TE buffer and store it under the cooling conditions.
Now you can use 5μl of our DNA ladder for any of the gel electrophoresis experiment.
If you are not comfortable with multiplexing the reaction (because more extraordinary expertise required to achieve good results in multiplex PCR),
Then perform the PCR reaction in 10 different tubes and load it on agarose gel along with the ready to use DNA ladder for conforming results.
The results of the PCR doing in 10 different separate reactions might look like this,
- A complete guide for analysing and interpreting gel electrophoresis results
- Part 2: Analysing and Interpreting (Agarose) Gel Electrophoresis Results
Producing our own 100bp or 1000bp (1kb) DNA ladder is a wise decision if the workload in the lab is very high.
The cost of the overall experiment for preparing the DNA ladder is much lower than the commercially available kits. Once you purchase primers, every time you can make your own DNA ladder.
I strongly believe that doing manual works like this improves our skills and expertise as well as reduces the cost of the experiment.
Wang, Tian-Yun et al. “Preparation of DNA ladder based on multiplex PCR technique.” Journal of nucleic acids vol. 2010 421803. 25 Jul. 2010, doi:10.4061/2010/421803