problems with dilutions: sensitivity - (Nov/07/2006 )
QUOTE (kukita @ Oct 12 2007, 02:05 AM)
Hi everybody! Sorry again for re-re-opening this topic 
... I feel better since Im not the only one having these dilution problems.
My standard curve with genomic DNA works fine, but lately my ten fold dilutions of cDNA are giving the same Ct... I've tried with a new synthetized cDNA, but this time the Ct was too much larger between ten fold dilutions than expected!! This way it is impossible to obtain consistent results
Please!! anyone knows why could this be happening? Maybe I have a nuclease or some other contaminant in my cDNA?? Or is it too saturated or too diluted to show the expected difference in the Cts?
Please please heeelp... I cant continue wasting more time on this!
Thanks a lot! kisses
... I feel better since Im not the only one having these dilution problems.My standard curve with genomic DNA works fine, but lately my ten fold dilutions of cDNA are giving the same Ct... I've tried with a new synthetized cDNA, but this time the Ct was too much larger between ten fold dilutions than expected!! This way it is impossible to obtain consistent results
Please!! anyone knows why could this be happening? Maybe I have a nuclease or some other contaminant in my cDNA?? Or is it too saturated or too diluted to show the expected difference in the Cts?
Please please heeelp... I cant continue wasting more time on this!
Thanks a lot! kisses
Hellooo guys:
I am here again, I was reading this old post and have to say that my problem was the small volume I was using for dilutions. Now I do dilutions of 3:27 (template:water). I calibrated my pippetes and always use the same, changing the tip each time and of course, using the vortex until my hand hurts a lot!!
-debokuki-
Thanks for the advice guys!!
Im gonna use different tips for every dilution, and start from 3 ul minimum... I was trying to save cDNA (sometimes I was using only 1 ul to start the dilutions) and using the same tip for all the serial dil... And of course always vortexing.
Wish me luck!
Thanks again 
kukita
-kukita-
QUOTE (kukita @ Oct 11 2007, 05:05 PM)
My standard curve with genomic DNA works fine, but lately my ten fold dilutions of cDNA are giving the same Ct... I've tried with a new synthetized cDNA, but this time the Ct was too much larger between ten fold dilutions than expected!! This way it is impossible to obtain consistent results
What source RNA are you using to make cDNA?
When I RT environmental RNA, I have to cesium clean it in order to rmove inhibitors that screw up the PCR (SYBR). Is your cDNA from envuironmental RNA?
-Patty4150-
Dear Patty4150,
Can you share you method of "cesium clean"?
thanks
-Hadrian-