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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