I add the β-Mercaptoethanol and DTT together with DNAZOL for my lysis buffer, sperm need harsh solution for open them. After washing with 70% ethanol 3-4 times. In the Nanodrop result, I still could see the 230 nm which are from the contaminant maybe guanidine thiocyanate (260/230), probably from the DNAZOL. If I use the QIAGEN kit, this 230 nm will totally disappear. But the problem for QIAGEN kit is very low yield of DNA when extracted from sperm, the columns maybe not available for large clumps of DNA to elute? Then, I have to use this old method to have high yield of DNA. I am concern this may affect my PCR reaction.
Hi,
Sorry for your contamination. TriZol based lysis buffers cause phenol carryover and cause 230nm absorption. Do not you add chloroform to generate phase difference? I do not know your protocol. There is a phase separation step by chloroform and centrifuge in the standard TriZol extraction.
Alternatively, I recommend Zymo RNA Extraction kit that is used for TriZol based RNA extraction without phenol carryover.
Good luck.
Hi, you can confront other protocols and tips and you can try to contact some author on researchgate
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