Hi,
I'm having difficulties with using qPCR to quantify my PCR-free libraries. There is just too much inconsistency in the end concentrations I obtain.
First of, does anybody do the two independent dilutions (1:10000, 1:20000) which Illumina recommends? Do you really dilute 2 µl in 20 ml and 40 ml?
When compared to the usual serial dilutions (1:1000, 1:2000, 1:4000, 1:8000), these two independent dilutions yield concentrations which aren't nearly similar to results from the serial dilutions.
E.g.
1:1000 -> 3.1 nM
1:2000 -> 4.2 nM
1:4000 -> 4.0 nM
1:8000 -> 4.2 nM
1:10000 -> 0.457 nM
1:20000 -> 0.397 nM
Another issue I'm having is a discrepancy between the first and other serial dilutions, in which the concentration is almost double what is obtained from the first dilution.
E.g.
1:1000 -> 4.5 nM
1:2000 -> 8.2 nM
1:4000 -> 8.2 nM
1:8000 -> 7.4 nM
What might I be doing wrong? Should I just take the results of dilutions which show at least some consistency (1:2000-1:8000) or should I include all serial ones (1:1000-1:8000)? What about the two independent dilutions which give weird results?
Thanks!
PS. I'm going to take a standard from the kit and try diluting it in the same manner to see if maybe my volume handling is the cause of these discrepancies between the dilutions.
I'm having difficulties with using qPCR to quantify my PCR-free libraries. There is just too much inconsistency in the end concentrations I obtain.
First of, does anybody do the two independent dilutions (1:10000, 1:20000) which Illumina recommends? Do you really dilute 2 µl in 20 ml and 40 ml?
When compared to the usual serial dilutions (1:1000, 1:2000, 1:4000, 1:8000), these two independent dilutions yield concentrations which aren't nearly similar to results from the serial dilutions.
E.g.
1:1000 -> 3.1 nM
1:2000 -> 4.2 nM
1:4000 -> 4.0 nM
1:8000 -> 4.2 nM
1:10000 -> 0.457 nM
1:20000 -> 0.397 nM
Another issue I'm having is a discrepancy between the first and other serial dilutions, in which the concentration is almost double what is obtained from the first dilution.
E.g.
1:1000 -> 4.5 nM
1:2000 -> 8.2 nM
1:4000 -> 8.2 nM
1:8000 -> 7.4 nM
What might I be doing wrong? Should I just take the results of dilutions which show at least some consistency (1:2000-1:8000) or should I include all serial ones (1:1000-1:8000)? What about the two independent dilutions which give weird results?
Thanks!
PS. I'm going to take a standard from the kit and try diluting it in the same manner to see if maybe my volume handling is the cause of these discrepancies between the dilutions.
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