PCR inhibitors - help ??!! (Dec/05/2007 )
what are the PCR inhibitors ????
can u give me a source,paper,book...
i know that ethanol is one of them..
buffers containing EDTA inhibit PCR.. cos it ll chelate magnesium needed for Taq activity!
too much phenol.. may change the pH or something.. not sure on this though!!
amount of DNA (too much)
animal fluids, food, organic soils, bacteria
forgetting to turn the machine on
ionic detergents (sodium deoxycholate, sarkosyl, SDS)
and the list continues.
I have tears streaming down my face.....
Note that most of these can be eliminated as issues by diluting your sample. Not quite to the level of homeopathy, you still do need a few molecules of DNA, but PCR is extremely powerful in amplifyiing, and dilution solves many inhibitor problems. You do still, however, need to turn the machine on and make sure it's plugged in.
not necessarily in that order.
i have read the following,
it has been suggested that calcium ions inhibit PCR by competing with the magnesium ions as a cofactor for the DNA polymerase.
The inhibition by EDTA may be related to its ability to inhibit DNA synthesis by chelating the Mg2+ necessary for the activity of DNA polymerase. Therefore, use of an increased magnesium ion concentration has been employed to maintain PCR activity in the presence of chelating agents.
Because of the ability of ethanol to precipitate phosphate
along with the DNA in the specimen, it was postulated that the
presence of phosphate in the final preparation was adversely
affecting the efficiency of the PCR. Increasing concentrations of sodium
phosphate in the samples progressively reduced the amount of
amplification product after 25 cycles of amplification.
where does the phosphate come from,knowing that DNA extraction was done by PCI method,proteinase k and SDS were added ???? is this its role in inhibition ???
The inhibitory effect of divalent ions (Ca and Mg) was more pronounced than that of monovalent ions (K and Na), with Ca being the most inhibitory.
how does monovalent inhibit taq polymerase activity ???
how does agarose,SDS inhibit PCR ???
thanks in advance..
sds will denature the polymerase.