nextgen life sciences

Products overview

Presented by Rahat Ali

Molecular biology : Molecular biology is the study of molecular underpinnings of the processes of replication, transcription, translation, and cell function.

The central dogma of molecular biology where genetic material is transcribed into RNA and then translated into protein, despite being an oversimplified picture of molecular biology, still provides a good starting point for understanding the field

DNA

RNA

Protein

DNA : DNA has double helix structure made of Base .

DNA Bases – A G T C

RNA : RNA is similar to DNA chemically but it usually has single strand.

RNA Bases- AGCU

Protein : Proteins are polypeptides( strings of amino acids)

A linear chain of amino acid residue is called polypeptide

Amino acid- a simple organic compound containing both a carboxyl (—COOH) and an amino (—NH2) group.

A protein contains at least one long polypeptide 50-100 residue

Short polypeptide 20-30 residue is commonly called peptide.

Expression cloning

Polymerase chain reaction (PCR)

Gel electrophoresis

Western blotting

• One of the most basic techniques of molecular biology to study protein function is expression cloning. In this technique, DNA coding for a protein of interest is cloned into a plasmid.

Vectors In molecular cloning, a vector is a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed. A vector containing foreign DNA is termed recombinant DNA. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Of these, the most commonly used vectors are plasmids. e.gGene art product portfolio from thermo fisher

Restriction Enzymes e.g Anza restriction enzyme cloning systems from thermo fisher

Custom DNA Oligos e.g Taqman Custom ologo synthesis service from thermo fisher

Competent Cells e.g One Shot® TOP10 Chemically Competent E. coli from thermo fisher

Microbial Culture Media for Bacteria, yeast and algae etc.

Transfection Reagents e.g Lipofectamine Reagents from thermo fisher

It is a molecular technology which aims to amplify a single or few copies of the DNA to thousands or millions of copies.

PCR targets and amplifies a specific region of a DNA strand.

It is an invitro technique to generate large quantities of a specified DNA.

Often, only a small amount of DNA is available eg.A drop of blood, Semen strains, Single hair, vaginal swabs etc.

NOTE : Two methods currently exist for amplifying the DNA or making copies◦ Cloning—takes a long time for enough clones to reach

maturity

◦ PCR—works on even a single molecule quickly

PCR Machine e.g proflex, simpliamp,veriti thermal cycler from thermo

fisher.

PCR plates, strips, tubes e.g ABI and abgene plastic

DNA Template

Primers

Taq polymerase e.g Dream Taq, Platinum taq, amplitaq fom thermo fisher

Deoxynucleoside

triphosphates(dNTPs)

Buffer solution

Ligation kit

PCR is highly versatile technique and has been modified in variety of

way to suit specific applications.

Inverse PCR

-In this method amplification of DNA of unknown sequence is carriedout from known sequence.

- This is especially useful in identifying flanking sequences of variousgenomic inserts

It is employed for amplification of RNA molecules .

-RT-PCR is widely used in expression profiling, to determine the

expression of a gene or to identify the sequence of an RNA transcript.

consumables for Reverse transcriptase PCR…

. cDNA synthesis kit

. Riverse transcriptase e.g super script product range

. RNase inhibiter

It is used to amplify and also for quantification and detection of DNA sample.

Real time PCR using DNA dyesFluorescent reporter probe method -Detection and quantitation of fluorescent reporter the

signal of which increases in direct proportion to theamount of PCR product in a reaction

-Does not measure the amount of end product but itsproduction in real time

Real time PCR and its consumables.

Quant studio Real time pcr product range from thermo fisher scientific.

Real time pcr is also called qPCR or quantitative PCR .

There are two common methods for detection of PCR products in real time PCR are…

1. Non specific fluorecent dye ( Dye Based)e.gsyber green dye or syber green mastermixes.

2. Sequence specific DNA probe ( Probe Based) e.g Taqman Probe mastermixes

Gel electrophoresis is one of the principal tools of molecular biology. The basic principle is that DNA, RNA, and proteins can all be separated by means of an electric field and size.

Requirements for Gel electrophoresis..

-Electrophoresis system e.g owl Gel Electro phoresis

systems

-Agarose gel

-Restriction enzymes

-DNA ladders

Western blotting

In western blotting, proteins are first separated by size, in a thin gel sandwiched between two glass plates in a technique known as SDS-PAGE(sodium dodecylsulfate polyacrylamide gel electrophoresis). The proteins in the gel are then transferred to a polyvinylidene fluoride (PVDF), nitrocellulose, nylon, or other support membrane.

◦ Requirements for western bloting◦ Gel Tank◦ Membranes ( PVDF,nitrocellulose, nylon)◦ Different western blot kit e.g west pico and ecl from Pierce

◦ Protein ladders.◦ Power supply

Molecular Biology

DNA

1. DNA Extraction 2. DNA isolation 3. gel Electrophoresis 4. Qubit Fluorimeter5. DNA ladders6. PCR- consists

products like..TaqDntp’sDyesBuffersLigation kitPlastic wares-pcr

plates, strips,tubes, seals.

RNA

1.RNA isolation kit e.gmirvana kit2. tryzol3. RNA later 4. cDNA – cloning

- Real time –sybrgreen and Taqman proBe5 Qubit fluorimeter

Protein

1.Protein purification-Ni-NTA resin2. Western bloting3.Protein ladders

4. Qubit fluorimeter5. Protein ladders

Media- Serum

DMEM FBS Heat inactivated

RPMI FBS one shot

IMDM FBS based on Origin

Gluta MAx

optiMEM