Basic DNA extraction question 3 - (Oct/09/2005 )
this is a flurry of questions to satisfy my insatiable curiosity:
-why do we pre-incubate pancreatic RNase at 85 degree and pronase at 37 degree?
-what is the role of the phenol in DNA extraction?
-why is a phenol/chloroform extraction preferable to a simple phenol extraction?
- after doing a electropheresis, how do you calculate the molecular weight?
if you the answer to any of these questions, do not hesitate to answer.
stuck on an exam or something?
It denatures proteins, then they get enriched at the phenol-water-interface so they can be removed
from the DNA-prep.
Phenol is still slightly soluble in water, so after the phenol-extraction the DNA-prep contains phenol
which inhibits enzymes very strongly by binding and denaturation. Chloroform-extraction removes
the phenol and can be easyly removed after EtOH-precipitation by drying.
You calibrate your run / gel by running a standard with DNA-fragments of known size along with
your samples, the rest works in the same way as vor TLC, Rf-values and that kind of stuff.
mol. wt can be measured by using a standard marker known as DNA Ladder. It is loaded in the first and last well then after running the gel we compare our dna fragments with the ladder. As the mol wt of ladder frgments are provided by the company we came to know the mol wt. of our fragment.