Gel electrophoresis

Question 1

what is Gel electrophoresis

Answer 1

is a method for separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments, based on their size and charge.

Question 2

how does it work

Answer 2

Gel electrophoresis apparatus – an agarose gel is placed in this buffer-filled box and an electrical current is applied via the power supply to the rear. The negative terminal is at the far end (black wire), so DNA migrates toward the positively charged anode (red wire).

Question 3

Gel electrophoresis is a method for separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments, based on their …………………

Answer 3

size and charge.

Question 4

sieving

Answer 4

is a phenomenon where Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel

Question 5

why is Proteins are separated by the charge in agarose?

Answer 5

because the pores of the gel are too small to sieve proteins

Question 6

role of gel

Answer 6

1. Gels suppress the thermal convection caused by the electric field application.
2. can also act as a sieving medium, and slow the passage of molecules.
3. gels can also serve to maintain the finished separation so that a post-electrophoresis stain can be applied.

Question 7

role of buffer in gel

Answer 7

1. used to provide ions that carry a current. ( These buffers have plenty of ions in them, which is necessary for the passage of electricity through them )
2. maintain the pH at a relatively constant value.

Question 8

Visualization

Answer 8

fter the electrophoresis is complete, the molecules in the gel can be stained to make them visible.

Question 9

Downstream processing

Answer 9

After separation, an additional separation method may then be used, such as isoelectric focusing or SDS-PAGE. The gel will then be physically cut, and the protein complexes extracted from each portion separately. Each extract may then be analyzed, such as by peptide mass fingerprinting or de novo peptide sequencing after in-gel digestion. This can provide a great deal of information about the identities of the proteins in a complex.

Question 10

application

Answer 10

Estimation of the size of DNA molecules following restriction enzyme digestion, e.g. in restriction mapping of cloned DNA.
Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting
Separation of restricted genomic DNA prior to Southern transfer, or of RNA prior to Northern transfer.

Gel electrophoresis is used in forensics, molecular biology, genetics, microbiology and biochemistry.