Lab Techniques

Question 0

PBS

Answer 0

Phosphate buffered saline - an isotonic solution that helps preserve the tonicity of cell and keeps sample at a physiological pH. Along with EDTA disingages attached cells

Question 1

EDTA

Answer 1

Chelating agent that binds metal ions such as MG which are cofactors of DNAses.

Question 2

SDS

Answer 2

Sodium Dodecyl Sulphate - detergent that lyses cells by solubilizing/denaturing lipids and proteins by disrupting non-covalent bonds. Increases proteinase K activity

Question 3

Proteinase K

Answer 3

Broad spectrum serine protease. Digests proteins. Therefore rapidly inactivating nucleases. Works well at a range of pHs and temps.

Question 4

Triton X-100

Answer 4

Detergent that solubilises membrane proteins and lipids.

Destroys mitochondria and organelles.

Question 5

Role of MgCl2 in PCR

Answer 5

Mg bind single nucleotide and acts as a cofactors for DNA polymerase.
Too much MG stabilizes dsDNA and prevents desaturation of strands.

Question 6

Tris

Answer 6

Primary role as buffer.

Also increases membrane permeability.

Question 7

Role of NaCl in DNA precipitation

Answer 7

Sodium neutralizes negative charge on DNA backbone making it less polar/hydrophilic. Preparing it for ethanol precipitation. If it remained negative is would stay in solution upon adding ethanol.

Question 8

Role of ethanol in DNA extraction

Answer 8

Ethanol has a lower dielectric constant (less polar than water) If enough ethanol is added attraction between sodium and phosphate backbone is strong enough for ionic bond to form and DNA to precipitate.

Question 9

Role of 70% ethanol

Answer 9

Wash Na molecules into solution whilst keeping DNA precipitated. It is ice-cold to prevent DNa denaturation.