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-   -   why are some dilutions of std curve detectble and some not? (http://www.molecularstation.com/forum/real-time-pcr-quantitative-pcr-forum/65358-why-some-dilutions-std-curve-detectble-some-not.html)

scitech 03-26-2009 10:37 AM

why are some dilutions of std curve detectble and some not?
 
I've made dilutions of my plasmid DNA for absolute quantification and it worked perfect when i used FAM-MGB probes from ABI, for the discrimiantion of biological samples to achieve real goal we had to change the design of primers and probes but my new set of FAM-TAMRA probes work with some dilutions of plasmid which i already had used for FAM-MGB and some dilutions do not give curves or some replicates of one dilution. can any body help to understand how to get rid of this

Aga 03-26-2009 12:03 PM

Re: why are some dilutions of std curve detectble and some not?
 
First important question is whether you used exactly the same dilutions or freshly made dilutions?

In the first case, if you freeze-stored your dilutions, there is a chance that some of the DNA degraded and this might have caused decrease in your DNA concentration.

With very low DNA copy number one or several of the replicates for one dilution might give negative results. The lower the copy number the higher the probability of obtaining such results.

If you prepared your dilutions just before the assay and obtained such results this may be caused by uneven dilution of your samples. It may be difficult to get identical DNA concentrations in all replicates if you have low concentration of your DNA in a certain dilution.

To make dilutions it is recommended to use rather larger volumes, if possible. So instead of making ten-fold dilution by adding 1ul of a sample to 9ul of buffer or water add 5ul to 45ul.

scitech 03-26-2009 02:57 PM

Re: why are some dilutions of std curve detectble and some not?
 
thanks for your reply, this is aliquot of same plasmid dilutions out of the freezer and just to rule out degradation of DNA, i made additional run with last set of primers and probes and it was o.k. as ever so DNA degradation or improper dilution is not reason behind...what else we can think of???

scitech 03-26-2009 03:09 PM

Re: why are some dilutions of std curve detectble and some not?
 
well this std dilutions are aliquots of older ones out of freezer and just to rule out degradation of DNA i had already tested it with old set of primers and probes and it was o.k. as ever...and yes my calculation is so that i use 5Ál of DNA per Rxn...so any other reaon which comes to your mind??????

scitech 03-30-2009 01:18 PM

Re: why are some dilutions of std curve detectble and some not?
 
i had counter checked with old primers and probes the DNA aliquots, there is no prob with DNA..i'm really confused how com it gives fluorescence in 30000000 copies and 3000 and not inbetween. one other question what if difference of synthesized oligos primers is 58 and tht of probe is 61 , will it work in case of FAM-TAMRA????????


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