9 Factors affecting DNA extraction from agarose gel

           Agarose gel electrophoresis is a method of choice for the identification, purification, and separation of the DNA fragments. DNA fragments from the gel are routinely extracted for various downstream processing. These include, cloning, radio-labeling, in vitro transcription, microinjection and sequencing of the DNA molecules.

            

           Gel extraction of DNA fragments is mainly done to remove proteins and salts that incorporate from certain reactions. Therefore, in order to use the DNA fragments for downstream processing, these components musts be removed. For example, a PCR amplification or restriction enzyme digestion reaction contains factors which inhibit further applications of the DNA fragment.

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Choosing the right DNA polymerase for your PCR

Success of the polymerase chain reaction (PCR) largely depends on the choice of the appropriate DNA polymerase. DNA polymerase is one of the major components needed for setting up a PCR. For PCR, a thermo-stable DNA polymerase is essential, so that it can endure higher temperatures during the cycling conditions. Therefore, thermo-stable DNA polymerases serve as a key player in the current methods of DNA amplification and sequencing.

Based on their amino acid sequences, DNA polymerases are categorized into six families: A, B, C, D, X and Y. Thermophilic enzymes are found in all the six families. Same reaction is catalyzed by all DNA polymerases, that is, adding nucleotides to the 3'-end of the DNA primer to synthesize the new DNA strand complementary to the template DNA. The thermo-stable DNA polymerases synthesize DNA in a template-directed manner, and require primer-template hybrid to begin the synthesis.

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Three commonly used affinity tags for protein purification

In order to purify heterologous proteins from various hosts, affinity tags are extremely capable tools. In the recent years affinity tags have become highly popular tools for protein purification mainly due to following reasons. They provide high level of purification of recombinant proteins from crude extracts, mostly in a single step. Second, they provide mild elution conditions, thereby, do not interfere with the structure and hence the function of the purified proteins. In addition, affinity tags allow a variety of proteins to be purified using easy procedures.

            Affinity tags are available as expression vector systems having multiple cloning sites (MCS) for cloning the gene of interest towards the N or C-terminal of the tag. Now-a-days, a variety of affinity tags are available for the purification of recombinant proteins. Each tag has its certain advantages and disadvantages. Here, you will see the properties of the three commonly used affinity tags: His-Tag, GST-Tag and MBP-Tag.

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RNA Isolation: Principle and Procedure

In order to perform various fundamental molecular biology experiments, the first and most crucial step is isolating high quality, intact RNA. These experiments include Northern hybridization, RT-PCR, RNA mapping, nuclease protection assays, in vitro translation, cDNA library construction and so on.

 In a cell, 80-85% of the total RNA is contributed by ribosomal RNA (rRNA). However, messenger RNA (mRNA) is only 1-5% of the total cellular RNA. 

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Extraction of DNA from agarose gel

The extraction of DNA molecules from agarose gels is routinely employed for the downstream processing of the DNA fragments. Gel extraction is frequently done for the cloning, radio-labeling and sequencing of the DNA molecules. Generally, the purification of DNA fragments involves the separation of DNA from agarose gel slices.

            The reaction components of certain molecular cloning techniques e.g. a PCR amplification or restriction enzyme digestion, include certain proteins and salts that may inhibit further applications of the DNA fragment of interest. Hence, in order to use the DNA fragments for additional purpose, these components musts be removed. It has been observed that purification of DNA generally results in the improvement of its efficiency in subsequent processing. For example, the digested DNA can be ligated and the PCR products can be directly cloned into the T/A cloning vector. 

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SDS-PAGE: Principle and Procedure

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) is the most desirable method used for the qualitative analysis of the protein mixtures. This method is basically used for checking purity of the proteins. Since, in SDS-PAGE the proteins get separated in accordance to their size, the method is also used to determine the molecular weight of the proteins. After performing the SDS-PAGE, subsequent specialized techniques such as Western blotting, two-dimensional gel electrophoresis and peptide mapping can be done.

With the help of SDS-PAGE, a wide range of proteins can be separated by preparing varying concentrations of polyacrylamide gels. Generally, 10% polyacrylamide concentration of gel is sufficient enough to resolve the proteins ranging from 10-150kDa.

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Five steps involved in the Expression and Purification of Proteins in E. coli

Production and detailed characterization of a variety of proteins is facilitated by their heterologous expression and purification. Recent advances in genomics have led to a massive increase in the number of proteins being produced using recombinant DNA technology. 

            

            In order to express heterologous proteins, a variety of expression systems have been developed. For example, bacteria (e.g. Escherichia coli, Bacillus subtilis, etc), yeasts (e.g. Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, etc), filamentous fungi (e.g. Aspergillus nidulance, Trichoderma reesei, etc), insects, plant cell cultures and mammalian cell lines. 

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Vectors: The Cloning Vehicles

The outstanding technique of gene cloning is largely dependent on vectors. Vector is a DNA molecule which is used for the transfer of foreign genetic material from one cell to another and, hence, also known as cloning vehicle. It has the capability to steadily maintain itself and replicate in an appropriate host organism.

A typical vector consists of the following components:

·         Origin of replication

The origin of replication present in the vector aids its efficient replication inside the host cells. During this process, the cloned DNA fragment in the vector gets replicated too. This results in the amplification of the cloned DNA fragment and hence any subsequent manipulations and analysis can be easily done. Moreover, the stored vector-DNA fragment construct can be further used to produce indefinite amounts of the DNA fragment.

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Five Factors influencing the migration of DNA fragments in agarose gel

            It is a well known fact that agarose gel electrophoresis of DNA is a routinely used method in molecular biology. The major applications of the technique includes: identification, purification, and separation of the DNA fragments. Frictional resistance caused by the gel matrix is responsible for the separation of the DNA fragments. As a result of this, the DNA fragments migrate in the gel according to their respective size.

            Passing through the gel pore becomes difficult for the larger DNA molecules whereas, the smaller DNA fragments migrate unhindered. This causes the DNA fragments to migrate according to their respective size. As expected the smallest molecule moves the fastest and the largest molecule moves the slowest.

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PCR Master Mix

            The polymerase chain reaction (PCR) is used to amplify a specific fragment of DNA strand from a complex mixture of initial starting material. For setting up a PCR, you need to prepare a master mix. Generally, the PCR master mix consists of a target double-stranded DNA template, two oligonucleotide primers which hybridize to bordering sequences on either strands of the template, all four deoxyribonucleoside triphosphates (dNTPs) and a DNA polymerase.

             Generally, you need to prepare 10-200µl reaction volume in small reaction tubes (200-500µl volume tubes) for setting up a PCR. The PCR is carried out in a thermal-cycler. Thermal-cycler is a machine that provides varying temperatures by heating or cooling the reaction tubes, as per the requirement of each reaction step. It is preferable if you use thin-walled reaction tubes, since it facilitates rapid thermal equilibration by maintaining proper thermal conductivity. 

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Gene Cloning: An Overview

Gene cloning is an outstanding molecular biology technique that has initiated a new era in the manipulation, analysis and exploitation of bio-molecules. Several discoveries and understandings of the gene structure, function and regulation are the significant outcome of the gene cloning process.

W. Sutton was the first person who proposed that the genes are located on chromosomes in 1903. Further, in 1910, this was proved experimentally by T.H. Morgan. Even though there was enough advancement in classical genetic studies, the actual revolution in the field of genetic research came with the discovery of the DNA as the genetic material. Avery, MacLeod and McCarty, in 1944, for the first time showed that genes are made up of DNA.  

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Agarose Gel Electrophoresis

             Agarose gel electrophoresis of DNA is a method routinely used in molecular biology to identify, purify, and separate DNA fragments. The mixed populations of DNA fragments are separated on the basis of their size.  The technique is easy as well as fast to perform. Moreover, agarose gel electrophoresis has good capability of resolving DNA fragments.

            It has been observed that DNA fragments ranging from 50bp to 20,000bp are well resolved using various concentrations of agarose gels. These characteristic features make this technique to take the centre stage in molecular cloning experiments.

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PCR

PCR is one of the most basic techniques having a major impact on various aspects of molecular biology. It provides massive and highly specific amplification of a precise fragment of DNA whose short sequences of either ends are known. Conceptually PCR is the oldest and practically it is the most versatile tool.

The concept of PCR dates back to the early seventies of the last century. In 1971, H.G. Khorana along with his colleagues, proposed a strategy for the chemical synthesis of the DNA. The method was the same as of now-a-days PCR. Unfortunately, due to lack of availability of many important factors it didn’t seem practical and hence was forgotten. 

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