5 Factors affecting gene cloning efficiency

Gene cloning is the central technique involved in recombinant DNA technology. Moreover, it facilitates the discoveries and understandings of the gene structure, function and regulation. A new era has been initiated as a result of this method in the manipulation, analysis and exploitation of bio-molecules.

However, competent gene cloning is not that much easy as it sounds. In order to efficiently clone the gene of interest into a particular vector, you need to be skilled. Since the efficiency of cloning is determined by several factors. Therefore, each factor should be deliberately considered to get best cloning efficiencies.

Here we provide you the most common factors which affect the efficiency of your gene cloning experiment.

1)         Starting Material

Well begun is half done. You know this notion very well. This applies to gene cloning also. A good starting material means you have done half of the things correct, only remaining half is to be optimized.

You materials should be pure. The isolated plasmid should be free from contaminating components. One of the most common contaminant is the media in which the culture is grown. This results in the poor digestion and ligation of the plasmid.

Therefore, during plasmid isolation, you should make sure that the media has been completely removed from the bacterial cells. Moreover, it is also a good practice to ethanol precipitate the plasmids prior to restriction digestion. This removes the salts present in the plasmid suspension and hence results in proper digestion of the plasmids.

If PCR product is the starting material then either reaction cleanup or gel extraction should be performed. We generally go for the gel elution of the PCR product as this not only removes the reaction components and primer dimers but also the non-specific amplifications.

2)         Digestion of the Vector and Insert

Digestion is very important factor determining the efficiency of the cloning experiment. Good cloning efficiency requires complete digestion of the insert and vector molecules. Digestion, if not properly done results in partial digestion of the vector and insert molecules, which in turn results in poor cloning efficiency.

However, it is relatively uncomplicated to achieve complete digestion. You should follow the stuffs quoted here in order to get desired results.

You should take units of the restriction enzyme according to the amount of plasmid or PCR fragment taken. Generally, 1-2µg of plasmid DNA/PCR product is a good amount for digestion. To digest this amount of DNA you should take 1-2µl (5-20 units) of the chosen restriction enzyme. Now-a-days many suppliers provide enzymes in a format that contains units in 1µl to digest 1µg of DNA. This really simplifies the digestion process.

Things become more complicated when you have to perform double digestion. While performing double digestion you should always check the buffer in which both the enzymes are having maximum activity. But there are certain enzymes which cannot be used for double digestion. The reason being the fact that there buffer requirements are not compatible. In that case it is better to perform sequential digestion.

However, sequential digestion results in the loss of the fragment so you will have to start with more amount of DNA. In addition, there are certain enzymes which have different optimum temperatures. In this case also you will have to go for sequential digestion.

However, there are certain suppliers (NEB, Fermentas etc) who have optimized a single buffer and a single temperature for a variety of commonly used enzymes. Unfortunately, these enzymes are little bit expensive but in our opinion are worth. Using such enzymes not only saves your time and energy but also improves the digestion many folds.

3)         Amount of digested vector to be taken for ligation

Well, after completely digesting the insert and the vector molecules, the question arises about the quantity of digested vector to be used for ligation. This significantly affects the efficiency of your gene cloning experiment.

The amount of vector to be taken solely depends on the size of the vector. For example, if the size of the vector is small then you will have to take little amount of vector and vice-versa.

We have made a generalized approach towards the size and amount of vector to be taken. According to our experiences, the optimum relation between size and amount of digested vector is as follows:

Size                                        Amount

<5 kb                                      50 ng

5-7.5 kb                                  75 ng

7.5-10 kb                                100 ng

> 10 kb                                   upto 150 ng

Taking amount of digested vector according to its size increases the ligation to a great extent and hence the cloning efficiency.

3)         Insert vs Vector Molar Ratio

Molar ratio plays a valuable role in the ligation of the fragments and hence in the cloning efficiency. But before moving further, let us see what molar ratio is? Molar ratio (sometimes also called as molar excess) is the amount of moles of insert per moles of vector molecule.

Generally, molar ratio is taken 3. That is, three insert fragments per vector molecule. It is a good practice to calculate the amount of insert in respect of vector and molar ratio prior to setting up the ligation reaction. The formula is:

ng of insert =  ^{amount of vector x molar ratio x size of insert} /size of the vector

For example, if the size of your vector is 6 kb and the size of insert is 1200 bp, then for setting up the ligation reaction you should take 75 ng of vector and 45 ng of insert.

However, if the difference between the size of insert and size of vector is more (e.g. insert=300 bp; vector=12000 bp), then molar ratio should be 5-10.

In order to accurately quantify the amount of eluted insert and vector molecules you should either use spectrophotometer or a nano-drop. However, we will not recommend you to estimate the amounts by running the samples in agarose gel. This does not allow accurate quantification of the fragment and hence results in lower ligation and cloning efficiency.

5)         Efficiency of the competent Cells

All well that ends well. This is also the case with gene cloning. If you have done all the things correct but you are not doing the last step properly, then you will ultimately ruin your whole hard work.

The ligation mix is used to transform E. coli competent cells and hence efficiency of the competent cells used significantly affects the cloning efficiency. We will recommend you to use high efficiency competent cells for your all cloning experiments. Always use the protocols which results in transformation efficiency of > 10⁷ cfu, for preparing competent cells.

However, if you are unable to prepare high efficiency competent cells, we will recommend you to go for commercially available competent cells. Nevertheless, you can easily prepare high efficiency competent cells, by going through our forthcoming article on competent cell preparation.

We hope that these informations will be helpful for your gene cloning experiments. However, if you have any further query regarding gene cloning, please feel free to post it in the comments.