Molbio Lab Flashcards (FINALS)

Electrophoresis

It is a physical method of analysis which involves separation of the compounds that are capable of acquiring electric charge in conducting electrodes

Opposite

Ions that are suspended between two electrodes tends to travel towards the electrodes that bears _______ charges.

Zone Electrophoresis

It involves the migration of the charged particle on the supporting media

• Paper

• Cellulose acetate membrane

• Starch Gel

• Polyacrylamide

Zone Electrophoresis

Advantages include:

• Useful in biochemical investigations.

• Small quantity of sample can be analyzed.

• Low Cost and easy maintenance

Gel Electrophoresis

Separation is brought about through molecular sieving (pores) technique, based on the molecular size of the substances

Porous gel

It acts as a sieve by retarding or, in some cases, by completely obstructing the movement of macromolecules while allowing smaller molecules to migrate freely

Agar

It is a mixture of polysaccharides extracted from seaweeds

Agarose

• It is a highly purified uncharged polysaccharide derived from agar.

• It is used to separate macromolecules such as nucleic acids, large proteins and protein complexes

Polyacrylamide gel (PAGE)

• It is prepared by polymerizing acryl amide monomers in the presence of methylene-bis-acrylamide to cross link the monomers.

• It is thermostable, transparent, strong and relatively chemically inert.

• Tougher than agarose gels

• They are run in non-denaturing conditions, so that the analyte's natural structure is maintained.

• Separation is based upon charge, size, and shape of macromolecules.

• Useful for separation or purification of mixture of proteins.

• This was the original mode of electrophoresis

Denatured-PAGE / SDS-PAGE

• Separation is based upon the molecular weight of proteins.

• The common method for determining the MW of proteins.

• Very useful for checking purity of protein samples.

Starch

• A suspension of ________ should be boiled in a buffer to give a clear colloidal suspension.

• The suspension on cooling sets as a semisolid gel due to intertwining of the branched chains of amylopectin.

• In order to avoid swelling and shrinking petroleum jelly is used

Starch

Advantages include:

• High resolving power and sharp zones are obtained.

• The components resolved can be recovered in reasonable yield especially proteins.

• Can be used for analytical as well as preparative electrophoresis

Thin Layer Electrophoresis

Studies can be carried out in thin layer of silica, alumina

Cellulose-Acetate Electrophoresis

• It contains 2-3 acetyl groups per glucose unit and its adsorption capacity is less than that of paper.

• It gives sharper bands.

• Provides a good background for staining glycoproteins.

Optical windows

CARTRIDGE COMPONENTS:

Enables real-time four-color detection

Reaction tube

CARTRIDGE COMPONENTS:

Thin chamber that enables very rapid thermal cycling

Valve

CARTRIDGE COMPONENTS:

Enables fluid transfer from chamber-to-chamber; may contain DNA lysis

Processing chamber

CARTRIDGE COMPONENTS:

Contains reagents, filters, and capture technologies necessary to extract, purify, and amplify target DNA

Mix, Inoculate, Load, Expect

What does MILE stand for in GeneXpert?

Sample Reagent Buffer

• It has decontaminating and digesting property to liquefy specimens prior to cartridge inoculation.

• Contains 2% sodium hydroxide and isopropanol

2:1 (2 parts buffer, 1 part specimen)

What is the buffer-to-specimen ratio?

TB Lamp

It is a unique, temperature-independent technique for amplifying DNA that is simple to use, providing a visual display that is easy to read

cDNA

It can be sequenced, cloned and applied to estimate the copy number of specific genes in order to characterize and to validate gene expression

Reverse Transcriptase

They are RNA dependent DNA polymerases which bind to RNA templates and convert them to their complementary DNA sequence in the presence of dNTPS and other essential cofactors

Dr. Howard Temin

The enzyme Reverse Transcriptase was first reported by

Short polypurine tract (PPT

It is relatively resistant to RNase H degradation

RNase H

It removes the primer tRNA, exposing sequences in +ssDNA that are complementary to sequences at or near the 3′end of plus-strand DNA

Reverse Transcription PCR

Developed to amplify RNA targets such as HIV, HCV, and influenza

It entails an initial step of transcribing a portion of the RNA genome into complementary DNA (cDNA) which is then amplified through PCR

Oligo dTs

They are synthetic DNA strands which can be represented as a sequence of ‘T’ nucleotides as in 5’ – G T T TT T TT TT T TT TT TT -3’

Gene specific primers

It can be designed to anneal to a specific gene

Random hexamers

They consist of a ten bases which anneal randomly to RNA molecules

RNA

It is single stranded and tends to form secondary structures via Watson-Crick base pairing