PCR & Western Blotting Flashcards
1
Q
Describe PCR
A
- Polymerase Chain Reaction
- used to amplify a sequence of DNA using pair of oligonucleotide primers, each complementary
- amplify a single copy or few copies of a segment of DNA across
2
Q
what are some essential components of PCR?
A
- template DNA that contains target sequence
- genomic (gDNA) or complementary DNA (cDNA)
- < 1ug of DNA is required
- thermostable DNA Polymerase = Taq polymerase
- pair of synthetic oligonucleotides to prime DNA synthesis
- buffer solution to maintain pH
3
Q
A
4
Q
What stages are involved in PCR ?
A
- Denaturation
- Annealing
- Extension/elongation
- Final extension
- Hold
5
Q
How many cycles does denaturation, annealing & extension have?
A
- 20-40 cycles
6
Q
How are proteins most ofen fractionated?
A
- by column chromatography, in which a mixture of protein in solution is passed through a column containing a porous solid matrix
7
Q
How can proteins be sorted?
A
- charge (ion-exchange chromatography)
- hydrophobicity
- size
- ability to bind to particular small molecules or to other macromolecule
