gDNA yield – Photometers do not tell the whole truth!
Before DNA is used for enzymatic analyses such as PCR, scientists often determine the total yield by spectrophotometry at 260 nm (OD260 value).
Short DNA fragments suggest a higher gDNA yield
DNA extracted using Silica kits (bind-wash-elute methods) contains small fragmented DNAs co-purified in significant amounts and also RNA, if no extra RNAse has been applied during the purification process. The resulting OD readings are misleading and indicate much higher amounts of genomic DNA than really obtained.
In the experiment described, DNA was extracted from 10 mg rat brain using a common Silica Kit (supplier Q) or the EchoLUTION Tissue DNA Micro Kit (4 samples each). Photometry indicates a 2-fold higher DNA yield using the Silica Kit (graph). However, if the same samples are loaded onto an agarose gel for analysis, it becomes obvious that the real gDNA content is a multiple lower with the Silica kit than with the EchoLUTION kit.
The same problem with RNA
Since the OD260 for RNA adds at least 25% more to the total amount measured than OD260 for DNA, the observed huge differences in DNA concentration calculated via photometry and compared to gel analysis can therefore easily be explained.
Therefore, in order to compare the yield of DNA preparations obtained from Silica and BioEcho DNA extraction kits, we recommend to always perform gel analysis or a functional assay such as qPCR.
Photometry: Misleading gDNA quantification with Silica bind-wash-elute kits –
correct with BioEcho single-step technology
Really the best yield with bind-wash-elute?
Due to its unspecific nucleic acid binding method, Silica kits purify any nucleic acid including small unwanted DNA molecules and also RNA, whereas the EchoLUTION technology extracts only the targeted high-molecular-weight DNA in high yields.
If scientists only rely on photometry, the gDNA yield will often be dramatically overestimated; downstream analyses such as PCR/qPCR or NGS will then suffer from low sensitivity and logically often fail.
Furthermore, Silica-based extractions are of lower purity, e. g., caused by carried-over small molecules that may inhibit enzymatic reactions – and even more so if input DNA concentration is too low! In contrast, EchoLUTION purified DNA is always free of inhibitors, contaminating RNA and small DNAs. Only these OD readings are reliable and represent the true DNA concentration and yield!
Only EchoLUTION removes unwanted small DNA and RNA
Consequently, applications downstream of a EchoLUTION DNA extraction tend to be much more sensitive and reliable than using bind-wash-elute DNA kits.
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