MOL BIO LAB MIDTERMS - ELECTROPHORESIS

• Arne Tiselius

Father of Electrophoresis

Negatively charged

DNA molecules has a charge of what?

Electric field

This type of field causes DNA migration toward the positive electrode

Moves faster

Smaller fragments moves at what pace through a gel matrix

Moves slower

Larger fragments move at what pace in a gel matrix

Agarose Gel Electrophoresis

A fundamental laboratory technique in molecular biology. Used to differentiate or separate DNA, RNA, or proteins by size.

Gel

This material acts as a "molecular sieve"

Gel

A colloid in a solid form (99%)

Neutral state

It is crucial for a support media to be in this kind of electrical state

Agar
Starch
Polyacrylamide gels

List the different types of gels

Agarose gel / Agar
Polyacrylamide gels

List the most commonly used gels

Separating DNA

Purpose of Gel electrophoresis

Agarose

Natural linear polymer extracted from seaweed. Forms gel matrix through hydrogen bonding.

Agarose Gel

A linear polysaccharide (made-up of repeat unit of agarobiose-alternating unit of galactose and 3,6- anhydrogalactose).

1% and 3%

Agarose gel is used at what concentration

Agarose concentration

Pore size is controlled by what

To separate DNA, proteins, HB variants, isoenzymes.

Application of Agarose gel

50 voltage

Voltage needed in a gel electrophoresis to separate molecules in 50 minutes

100 voltage

Voltage needed in a gel electrophoresis to separate molecules in 30 minutes

120 voltage

Voltage needed in a gel electrophoresis to separate molecules in 25 minutes

Gel casting trays and gel casting stand

This equipment come in variety of sizes and composed of UV transparent plastic.

Sample combs

This where molten medium is poured to form sample wells in the gel.

Tris-acetate-EDTA and Tris-borate-EDTA

Mostly used electrophoresis buffer

Lithium Borate-Buffer (8.6 pH)

Highest pH value in a electrophoresis buffer

Loading buffer

This type of buffer contains something dense to allow the sample to "fall" into the sample wells, and one or two tracking dyes.

Glycerol

This substance is used in a loading buffer to allow the sample to fall into the sample wells

Bromophenol blue or Xylene cyanol

This substance is used in a loading buffer that migrates in the gel and allow visual monitoring or how far the electrophoresis has proceeded

Ethidium bromide

DNA molecules are easily visualized under an ultraviolet lamp when electrophoresed in the presence of this substance although it is toxic and carcinogenic.

Gel red and SYBR green

Substitute staining substances for ethidium bromide

DNA ladder

Reference marker or guide to know what is the size of DNA base pairs.

Transilluminator

An ultraviolet light box. This is used to visualize stained DNA in gels.

0.3% to 0.7%

What is the percentage of lower concentration in agarose to have large pore (5kb to 60kb)

0.7% to 2%

What is the percentage of medium concentration in agarose to have medium pore (0.8 to 10Kb).

2% to 3%

What is the percentage of high concentration in agarose to have small pore (<1kb)

Low concentration

The aim is to separate large DNA fragment what should be the concentration of agarose

High concentration

if the aim is to separate small DNA fragment what should be the concentration of agarose.

Preparation of Agarose Gel
Sample Preparation
Loading Buffer Components
Running the Gel

Steps involved in Agarose Gel Electrophoresis

Simple and rapid preparation

Cost-effective

Wide range of separation

Non-denaturing conditions

DNA recovery possible

High resolution for specific size ranges

Advantages of Agarose Gel Electrophoresis

Gel melting at high voltage

Buffer depletion

Uneven migration

Poor resolution

Band smearing

Contamination tissues

Limitations and Troubleshooting of Agarose Gel Electrophoresis

Paper Electrophoresis

Gel Electrophoresis

Cellulose acetate Electrophoresis

Isoelectric Focusing

Immunoelectrophoresis

Types of Zone Electrophoresis

Capillary Electrophoresis

Isotachophoresis

Types of Moving Boundary Electrophoresis