I extracted whole blood samples using the Qiagen DNeasy blood and tissue kit. When quantifying the DNA on the Qubit, I get baffling low DNA concentrations. Is there something that I might be doing wrong? Is there a way to increase my DNA yield after extractions?
Thank you!
Dear Sonja,
What Qubit kit you use for DNA measurement?
The HS kit is suitable for low concentration DNA solutions.
Try to input 10 uL of your DNA solution in 190 uL test mix.
All the best.
Hi Vladimir,
Thank you for the answer. I am using the HS assay. Regardless of the input, the stock calculation still shows the same low concentrations.
Hi Sonja,
I haven`t expirience of DNA isolation with Qiagen DNeasy blood and tissue kit.
I found in the Handbook "In order to obtain optimum DNA yield and quality, it is important not to overload the DNeasy spin column or DNeasy 96 plate, as this can lead to significantly lower yields than expected (see Figure 1)." Figure 1 is very strange.
Try to use appropriate blood volume. Try to use other manufacturer DNA isolation kit.
And you must be sure that your blood samples are not leukopenic.
Hello Sonja,
I have extracted a lot of fresh human blood samples using the QIAMP DNA blood mini kit from QIAGEN (cat 51106 250 samples). I use 200 -300 uL of blood and elute in 40 uL of buffer. The DNA concentration with Qubit dsDNA broad range assay kit (cat Q3850 Life Techn) ranges from 40 - 400 ng/uL. When I use buffy coat also I get from 40 - 800 ng/uL depending of each sample. You need to consider a couple of things: 1. The extraction kit you use is for certain species (you do not mention blood of what species you are extracting), the genomic DNA size depend of the species and also the type of column every kit is intended for. 2. The DNA yield depends of what is the starting volume of blood you are using, you cannot overload the column.3. When you said you get a low concentration is based on your assumption or your are comparing with another DNA reading method as Nanodrop, remember the readings with QUBIT are much less that with Nanodrop (for blood is probably 3 fold less than with Nanodrop) this is because the Qubit is reading intact DNA and it is very specific just to read good quality of DNA. I hope this help.
Cecilia Santrich
Hi Cecilia,
Thank you so much for the detailed explanation. :)
Kind regards
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