plasmid DNA dilution for absolute quantitation - (Apr/21/2006 )
i was advised to dilute my plasmid DNA for absolute std curve construction for gene expresion study using 0.2 microgram/microlitre low molecular weight sperm xenoDNA as the diluent. does anyone knows what is the purpose of this xeno DNA?
besides, does TRIS HCL buffered water can become one of the real time PCR inhibitors?
my plasmid do not amplify when i follow this protocol. I am using SYBR Green detection. thanx alotsss....
tris buffer may be inhitor if the quantity of plasmid added in proportion o total reaction solution is too big. The pH of RTPCR may be different than the trishcl solution for stocking and may decrease efficiency. But if pH are the same, i don't see why it could be inhibitor.
Of course EDTA should be avoided.
Regarding xeno DNA : maybe it acts as a DNAse preventing agent in sense that the total number of DNA molecules targetable by DNAses would be greater. Then your plasmid would less be affected. (kind of DNAse titration...)
The foreign DNA also acts as a blocking agent for glass or plastic sidewalls, reducing the amount of your target DNA taken out of solution on those surfaces. This is particularly a problem with very low DNA concentrations of your target, as found in the QPCR standards you are making.