using sybergreen to quantify DNA and assess its quality - (May/15/2013 )
Hallo everybody, I am doing seqeuncing for several bacterial samples,so i am extracting DNA using promega then am checking gel intensity and integrity by using agarose gel and ethedium bromide. then i am sunjecting the good sharp bands DNA (as i saw them on gel) to PCR to see the 16s amplification. the problem is that my supervisor said to me why do u waste time to check genomic DNA on gel with ethedium bromide , go and use syber green with RT-PCR it will show you the purity and concentration of DNA no need for gel. actualy i Don't know how to do his way and i am not that much expert in this feild. anyone can tell me the procedure of doing his way?? beside i got some non spesific binding in the first step of pcr , is increasing temperature could help to remove this non spesific binding? and how much should i increase??
SYBR green and RT-PCR (presuming by this you mean real-time- or qPCR rather than reverse transcriptase-PCR) will not tell you the concentration or integrity of the DNA - it can, if used appropriately, tell you the number of copies of a genome or specific DNA (or cDNA) sequence that you have in a sample, which then can be related to the concentration. Concentration of a sample can easily be assessed by using a spectrophotometer and measuring the absorbance at 260 and 280 nm.
Integrity of the DNA or RNA can only be assessed by running on a gel or a gel like chip (e.g. the Agilent gel on chip system).