I am working for a small CRO which hopes to validate my qPCR method prior to purchasing a qPCR machine. I have attempted to validate my method using a conventional PCR machine while measuring fluorescence every 8-10 cycles on a plate reader. In order to do so, I have to transfer the samples from a PCR plate into a clear-bottom plate as well as allow the samples to reach room temperature. The results were inconclusive. Furthermore, the plate reader had difficulty reading the small sample volume, and the numerous transfers led to loss of sample which further hurt my results.
I do get a small SYBR Green signal for the first 20 cycles, or so, but then amplification appears to halt.
I tried to use a nanodrop one instead of the plate reader. However, the oligo primers interfere with the dsDNA measurement. Any ideas? Thanks so much!
I don't understand why are you doing such hectic task that will definitely lead you nowhere.
The issues with conventional PCR, you are performing may be:
When you take plate out and bring it to room temperature for reading in plate reader and replacing in PCR without initial Taq activation temperature with time (As Taq need to be activated at recommended 94-98 degree C for 8-10 minutes to start work), it should not restart, so no further reading afterwards.
If your plate reader can read the fluorescence of try this way;
Use PCR stipes rather than PLate and set up at least triplicate reaction (exactly same) and bring 1 strip at one strip at different time points. Transfer the reaction mix to a plate that can be read.
that way your reaction set up will not be disturbed.
Hope this work.
I don't understand why are you doing such hectic task that will definitely lead you nowhere.
The issues with conventional PCR, you are performing may be:
When you take plate out and bring it to room temperature for reading in plate reader and replacing in PCR without initial Taq activation temperature with time (As Taq need to be activated at recommended 94-98 degree C for 8-10 minutes to start work), it should not restart, so no further reading afterwards.
If your plate reader can read the fluorescence of try this way;
Use PCR stipes rather than PLate and set up at least triplicate reaction (exactly same) and bring 1 strip at one strip at different time points. Transfer the reaction mix to a plate that can be read.
that way your reaction set up will not be disturbed.
Hope this work.
Hi Amber,
I do not recommend performing the qPCR using a conventional thermal cycler because of the problems you reported: poor precision and low sensitivity. In addition, manual pipetting steps will affect the final sample concentration, increasing the quantification inaccuracy. Quantifying on agarose gel is also very inaccurate, you may only be able to see a difference with a 3-5 fold change, which means you'll miss the first few cycles.
In my hands, this kind of "poor man's qPCR" works fine using agarose gel electrophoresis. I stained with gel red, which is extremely sensitive. And I used multiple reactions, each of which gets sacrificed to make a time-point. So I don't need to draw down individual reaction volumes over time.
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