PCR and Gel electrophoresis

What is the goal of gel electrophoresis?

Separate biomolecules (DNA, RNA, proteins) for analysis or purification

How are samples loaded into a gel?

Into wells (indentations) at the NEGATIVE electrode side, then an electrical current is applied

Why do DNA and RNA migrate toward the positive electrode?

Their phosphate groups carry negative charges, which are attracted to the positive electrode

What determines how far DNA/RNA fragments move in the gel?

Size. Smaller fragments move farther and faster through the gel pores.

How do you interpret bands in DNA gels?

Largest fragments stay near the wells (top), smallest migrate to the bottom. Bands represent fragment sizes; a ladder/marker provides reference (1400 bp, 300, etc)

What does one band in a lane mean?

The sample contains one fragment/component of DNA

How are proteins analyzed in gels?

Proteins denatured, disulfide bonds broken, and coated with SDS detergent to give uniform negative charge

What happens to proteins during SDS-PAGE?

They are boiled, unfolded, and destroyed. PURELY analytical, no purification

Under SDS PAGE conditions, proteins are separated based on…

Size (smaller proteins migrate farther)

Which biomolecules can be purified using gels?

DNA (proteins are destroyed in SDS PAGE)

What does PCR stand for

Polymerase chain reaction

What is the goal of PCR

To make many copies of a specific DNA region (not whole chromosomes)

What components are needed in a PCR tube?

Template DNA

DNA Primers

DNTPs

Heat stable DNA polymerase (Taq)

Buffer (Mg2+)

Mneuomonic: Toilet Paper does take bucks.

Toilet: Template DNA

Paper: Primers (DNA)

Does: dNTPs

Take: Taq (heat stable DNA polymerase)

Bucks: Buffer (Mg2+)

Why is helicase NOT needed in PCR?

Heat (95 degrees C) denatures DNA strands, replacing helicase

Why is ligase not needed in PCR?

Primers are DNA and remain in product, no Okazaki fragments to seal

What are the three stages to PCR?

DENATURATION (95 C), separate parental strands

ANNEALING (55 C) Primers base pair to target region

EXTENSION (72 C): Taq polymerase extends from primers 3’ end

Dont get me started on 95

Any 55 year old can navigate the highway

Even 72 year olds

Why is Taq polymerase used?

It comes from thermos aquaticus, a heat tolerant bacterium, withstands high temps

What is the optimal extension temperature for Taq polymerase?

72 C

How many copies are made after 30 cycles of PCR?

2 ^ 30, about a billion.

Why are two primers required for PCR?

One for each strand, pointing toward the region of interest, to enable exponential amplification

What happens if only ONE primer is used in PCR?

Linear amplification (not exponential), 30 rounds=30 copies DNA

Do PCR primers get removed from the product?

No, they are DNA primers and remain in the product