Electrophoresis

1. Not enough agarose added

2. Not heated enough to dissolve agarose

Problem: Gel doesn’t solidify

Gel wasn’t cool when the comb was removed

Problem: Collapsed well

Agarose leaked past the dams. Put the dams in securely

Skinny gel or irregular gel

Agarose leaked past the dams. Put the dams in securely

Problem: Well too small (sample won’t fit in the well)

50x TAE not diluted correctly

Problem: Gel runs too slowly

little or no 50x TAE added to electrophoresis buffer

Problem: Gel runs too quickly

1. Poor DNA Purification (e.g. minipreps)

2. sample leaked out of the well

Problem: Only the ladder is visible under the UV light

ELECTROPHORESIS OF NUCLEIC ACIDS

it is very useful, especially in molecular laboratories, if you wanted to detect the presence of certain substances such as protein and nucleic acids.It involves separating nucleic acids by size using an electric field, which is crucial for various molecular biology applications.

ELECTROPHORESIS

• will be used to separate and to analyze certain substances

• Defined as the movement of molecules by an electric current

ELECTROPHORESIS

• Routinely applied to the analysis of nucleic acids and proteins

• Under an electric current, DNA and RNA will migrate toward the positive pole

• it could occur in solution or we can use a matrix or solid gel

phosphate group

our nucleic acids are negatively charged because of their ______??

MIGRATION OF NUCLEIC ACIDS

DNA and RNA migrate at speeds inversely related to the size or length of the polymer

MIGRATION OF NUCLEIC ACIDS

Could be performed in tubes, slab gels, or capillaries

tubes, slab gels, capillaries

We have different types of polymers we can use for the migration of the nucleic acid:

GEL SYSTEMS

it will provide resistance to the movement of the molecules under the force of an electric current

GEL SYSTEMS

Serves as a support medium for analysis of the separated components

Unaffected by electrophoresis

Simple to prepare

Amenable to modification

Characteristics of a good matrix:

AGAROSE GEL

Polysaccharide polymer extracted from seaweed

(100 to 300 nm)

The concentration of the agarose dictates the size of the spaces in the gel

50-500 base pairs [2%-3%]

Small pieces of DNA

2000-50,000 base pairs [0.5%-1%]

Larger fragments of DNA

below 0.5% agarose concentration

are not practical [weak gel; easily broken]

will just impede the migration; highly be impeded

agarose gel

it is a natural polymer.

Agaropectin

The composition of the agarose gel or the polymer that can be extracted from the seaweed is called???

Agarose gel in solution

Powdered form of the agarose

Agarose gel comes into two (2) forms:

Agarose gel in solution

• just pour it directly onto the molds or culture media

• No need to prepare for specific concentration. No need to compute for the amount of the powder

Powdered form of the agarose

• manually you have to determine the amount needed and the amount of water na pang-dissolve sa powder

• this is the form of agarose gel we use in the laboratory

PULSED-FIELD GEL ELECTROPHORESIS [PFGE]

Modification of agarose gel electrophoresis

PULSED-FIELD GEL ELECTROPHORESIS [PFGE]

Instead of making a straight line of the migration of DNA, here, the migration is in a different direction so that the length of migration will increase so we will be able to separate large fragments.

PULSED-FIELD GEL ELECTROPHORESIS [PFGE]

• Current or voltage is applied in alternating orientation

• It is used if you want to type certain bacteria and do epidemiologic study.

conventional electrophoresis

the nucleic acids will migrate or move in a straight line or manner

Field-Inversion Gel Electrophoresis (FIGE)

Transverse alternating-field electrophoresis (TAFE)

Rotating Gel Electrophoresis (RGE)

Contour-clamped Homogenous Electric Field (CHEF)

TYPES OF PFGE

Field-Inversion Gel Electrophoresis (FIGE)

• This is the only one that moves 180°

• Periodically, it moves forward and backward

Field-Inversion Gel Electrophoresis (FIGE)

• Once electricity is applied, it will move towards the positive pole; then, mago-off/magpapalit. Tendency, babalik ‘yung nucleic acid

• There is alternation between the postive and negative pole [atras-abante]

Transverse alternating-field electrophoresis (TAFE)

• Moves 120°

• We have two sets of electrodes (1 top and bottom, 1 on the sides)

• There is alternating migration (top, bottom, and sides) – zigzag [mas nagiging mahaba ang path. In this way, we can separate the nucleic acids]

Rotating Gel Electrophoresis (RGE)

• Moves 120°

• Moves from side to side

Contour-clamped Homogenous Electric Field (CHEF)

• Moves 120°

• Just like TAFE, but instead of top, bottom, and sides, nasa corner ang dalawang sets ng nucleic acids.

POLYACRYLAMIDE GELS

Synthetic material

- free to do modification or to change the composition

POLYACRYLAMIDE GELS

Best for very small DNA fragments, single-stranded DNA, RNA, and proteins

POLYACRYLAMIDE GELS

Used for nucleic acid sequencing, mutation analyses, nuclease protection assays, and other applications requiring high resolution of nucleic acids

POLYACRYLAMIDE GELS

Acrylamide + Methylene Bisacrylamide

POLYACRYLAMIDE GELS

This is our crosslinker. So we have the crosslinker and we have the monomer

POLYACRYLAMIDE GELS

it is toxic. Particularly, it is neurotoxic – especially the monomer, acrylamide.

Polyacrylamide Gel Electrophoresis

PAGE

POLYACRYLAMIDE GELS

it could separate even if there’s a single base change - it can detect even one base pair difference in every 1,000 base pair

POLYACRYLAMIDE GELS

Usually done on protein analysis, aside from nucleic acids

POLYACRYLAMIDE GELS

it can give us a precise control of the properties; it is also used for mutation analysis

powdered form

most carcinogenic or neurotoxic - it is neurotoxic because it contains the monomer acrylamide

CAPILLARY ELECTROPHORESIS

• Automated version of electrophoresis

• We can use a solution that will serve as the medium; no need for gels; we use capillary tubing

• Usually used by large companies, etc.

CAPILLARY ELECTROPHORESIS

Used for the separation of organic chemicals such as pharmaceuticals and carbohydrates and also applied to the separation of inorganic anions and metal ions.

CAPILLARY ELECTROPHORESIS

also used for the separation and analysis of nucleic acids.

Fused silica

• Thin glass capillary

• Preferred since it is the thinnest and transparent material that will allow the passage of the fluorescent light [we use laser here to detect the molecules]

CAPILLARY ELECTROPHORESIS

• Separation was based on size and charge (charge/mass ratio)

• The smaller the molecules, the negative charge the molecules are, the faster the migration is

Fluorescent labels are covalently attached to nucleic acids to be separated by

CAPILLARY ELECTROPHORESIS

Used extensively in forensic applications and parentage testing performed by analyzing short DNA fragments

electrokinetic, hydrostatic, or pneumatic injection

The sample goes into the capillary through three injections:

CAPILLARY ELECTROPHORESIS

It is fully automated – the machine itself will analyze the molecule to be detected [medtech – interpret the results only]

BUFFER SYSTEMS

The purpose of a buffer system is to carry the electric charges or the current and protect the samples during electrophoresis

buffer

is a solution of a weak acid and its conjugate base

buffer solution

should remain constant; as the buffer molecules takes up or release proteins, dapat taga-carry lang siya ng charges, without affecting the result

BUFFER SYSTEMS

Control of the pH of a gel by the buffer protects sample molecules from damage - Near the neutral pH

Tris borate EDTA

• Preferred because they remain partly uncharged

• So, even on the desired pH po, they will remain uncharged.

Tris borate EDTA

The disadvantage is it is prone to precipitation

Tris acetate EDTA

• it's faster

• Disadvantage: easily exhausted especially if high voltage of electrophoresis or extended electrophoresis is used.

BUFFER ADDITIVES

modify sample molecules in ways that affect their migration

BUFFER ADDITIVES

So these are being added on our buffer system to inhibit the formation of the other secondary products

BUFFER ADDITIVES

These are denaturing agents used to remove the hydrogen bonding

Formamide

We add heat to ensure that there will be no occurrence of re-annealing

Formamide

by preventing the hydrogen bonding of the DNA, it blocks the hydrogen binding sites of the complementary sequences from re-annealing to maintain very long and straight DNA chains

Urea

The purpose of urea and detergent, it prevent formation of secondary structures such as single-stranded nucleic acid

Urea

It is used to prevent intrastrand homology – hairpin-like structures

pH DRIFT

There is an increasing amount of electrodes on both of the side of our electrophoresis

pH DRIFT

Decrease of pH at the cathode [–] and an increase of the pH at the anode [+]

Peristaltic Pump

To prevent pH drift, we have to move some of the buffers from the cathode going to anode using this ____?

HORIZONTAL GELS

Also known as the submarine gel

HORIZONTAL GELS

Cast as square or rectangular slabs of varying size