I'd like to use BioRad's iScript cDNA Synthesis Kit to make cDNA for qPCR. At the end of my current RNA extraction protocol I suspend the RNA in TE buffer (10mM Tris 1mM EDTA; pH 8.0). Will the EDTA present in the TE buffer interfere with later cDNA synthesis?
The iScript kit allows for 15 ul of RNA in a 20 ul reaction volume. My samples are relatively low concentration (40-70 ng/ul), so I will need to use most of the 15 ul. This means the 1mM EDTA will not be diluted much. I have already confirmed that the RNA is of good quality/purity.
Given the situation you describe, it's likely that ~1.0 mM might interfere with the cDNA synthesis. To get around it, you can increase the cDNA reaction volume up to 50 uL and add extra Magnesium to be on the safe side. Please do not heat up (65 deg) your RNA in the presence of Mg as it might degrade. Any heating to open up secondary structure has to be done prior to Mg addition. Most recent protocols don't heat up especially if Random Primers are used.
after extracting RNA, please elute with the elution buffer that came with the kit or with nuclease free water. if the buffer did not come with the kit, i strongly suggest that you dont elute your RNA in it as RNAase are present every where and it is very easy to degrade your samples. good luck with your work.
Thanks everyone, sounds like it might be safest to just elute with water.
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