How to do research in biotechnolgy – Qiagen, Thermo Fisher Scientific etc.

In this article, you will find a road map of biotechnology research.I used many kits, but here just for example, I’m using Qiagen, Thermo Fisher Scientific, and nucleopore kits.There ae many more companies that provide good kits.

The primary task in biotechnology it to isolate a gene and make its clone. For cloning the selection of the vector depends on the organism of interest.If interested in the expression of the gene, we use expression vector. Otherwise, we use multiplication vector.

I’m only giving a generalize idea without focusing core research.Steps may change depending upon the aim of the experiment.

The basic steps are:-

  1. Isolation of Nucleic acid from the sample.
  2. Amplify the gene of interest.
  3. Clean up the amplified sequence
  4. Ligate the purified sequence with the vector.
  5. Do transformation using competent cells.
  6. Select the positive clone.
  7. Overnight culture positive clone
  8. Plasmid isolation from transform cells
  9.  confirm the presence insert by restriction digestion.
  10. Preserve transform cells in glycerol stock.
  11. Bioinformatics analysis

 

Isolation of Nucleic acid from the sample

In this step, we isolate DNA or RNA from the sample by using kit based methods or manual methods.Electrophoresis does the qualitative and quantitive analysis.There are many different methods of nucleic acid isolation depends upon the type of sample going to use.For example,

  •  Isolation of DNA from Plant: DNeasy Plant Mini Kit – QIAGEN
  • Isolation of RNA fromPlants: RNeasy Plant Mini Kit – QIAGEN

Amplify the gene of interest

The particular gene is amplified from DNA and RNA by doing PCR or reverse transcription respectively.

  • PCR amplification with – DreamTaq Green PCR Master Mix (2X) – Thermo Fisher Scientific
  • For amplification of virus – REPLI-g Mini Kit – QIAGEN

Clean up the amplified sequence

Gel electrophoresis checks the amplified sequence.If it shows single amplified band, it’s good. We can use it for ligation with vector, but it is preferred to clean up PCR product. The salts present in PCR reaction mixture interfere in ligation process.

In the case, if you see more than one band in the gel: identify the right band, cut it from the gel and do gel cleanup.

  • QIAquick PCR Purification Kit – Qiagen
  • SureExtract Spin PCR Clean-up/Gel Extraction Kit

Ligate the purified sequence with the vector

In this step, our gene or desired sequence ligated with the vector. It depends on you what vector you are using.

  • Rapid DNA Ligation Kit – by Thermo Fisher Scientific

Transformation

In this step, our vector with the insert is transferred inside the bacteriaL cell for multiplication.Most of the time DH5α strain of E.coli is used.These transformed cells are cultured in nutrient media with a particular antibiotic.Only the transform cell survive in the presence o the antibiotic.

  • TransformAid Bacterial Transformation Kit – Thermo Fisher Scientific

Selection of the positive clone

Finding the correct positive clone is important.For this, we do colony PCR with the specific primer pair with respect the to vector.

The positive colonies are cultured overnight to increase the number in the presence of the antibiotic.

  • Rapid DNA Ligation Kit – by Thermo Fisher Scientific

Plasmid isolation

The plasmid is isolated from the overnight culture of positive colonies. These plasmids are utilized in different ways to perform more experiments.

  • SureSpin Plasmid mini kit – Genetix Biotech

Confirming the insert by restriction digestion

The isolated plasmids are checked for the insert by doing restriction digestion. The plasmid is digested with a particular enzyme and checked in agarose gel for confirmation

  • Restriction Enzymes – Genetix Biotech

Bioinformatics Analysis

In most of the times, the PCR amplified product or plasmid are sequenced to do further analysis. It involves sequence comparison, annotation, preparation of phylogenetic tree, docking studies, etc.

Visit these websites for bioinformatics analysis:-

NCBI – National Center for Biotechnology Information

EMBL – European Molecular Biology Laboratory

DDBJ – DNA Data Bank of Japan

Some companies links

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