pcr - etbr & dna extract (Nov/07/2005 )
1. can somebody tell me as to how much of DNA will be detectable on etbr containig gel (the lowest limit detectable under routine conditions)
2. i also need someone who can interpret the following results of a DNA sample which was diluted 1:200 -
UV spectrophotometry readings-
OD 260- 0.0036
OD 280- 0.0004
OD 260/ OD 280 = 9.0
from the same DNA extract when 5 microlitres of it was ran at a different occassion on a 0.8% gel but no band was seen.
should it have been detectable or not? can we treat the above values as correct? or can any of them be relied completely or none of them can be relied upon? is there any possible explanation to above findings including possible errors?
what is the dilution of your sample?
if no dilution : DNA conc would be : 0,18ng/µl. So 5µl are 0,9ng. That is undetectable on gel. "correct" minimum is 10ng.
By the way your OD is very very low. i think that a good measurment (for further exploitation) should not be less than 0.1
A 9 ratio don't tell anything at this level of OD... try to analyze the blank and you'll probably get the same OD or more...
For view DNA in EtBr Stain u should load ~ 18-20 ng of DNA