Hi all!
I can't dive into details, but I'm having issues with neutralising sodium citrate pH 5 in a 2-step cDNA synthesis and final RT-PCR. I'm manipulating my reverse transcriptase (100uL volumes) and need to add 2.5uL 1M sodium citrate pH 5 to finalise my solution.
Obviously, RT-PCR is quite sensitive to pH range, so I've been attempting to neutralise the solution prior to second step cDNA synthesis with 0.5M NaOH. However, I can't seem to reproduce any of my data when measured in RT-PCR (even when I titrate out my sample to investigate)
Any advice? Is the pH the main issue or is sodium citrate just not applicable to cDNA synthesis and RT-PCR? Is the sodium citrate impacting my RNA if the sample is too low of a concentration?
Any help or ideas are appreciated
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