I am having negative results for my DNA/RNA extraction from stool samples on Nanodrop for Concentration, 260/280 and 260/230. This is the first time I'm having this problem and have tried to use the blank with other samples, it works fine. When I tried on a new blank, still the same negative reading but diff value. I am using QIAGEN Allprep extraction kits. Anyone have any advises?
Which protocol did you follow for DNA extraction? This attached protocol of DNA extraction from stool might help
Best regards,
Mohamed
According to the attached bulletin (p.26):
Q: What is the cause of negative absorbance values?
A: A blank measurement was made on a dirty pedestal or using a solution with more absorbance than the sample of interest. Clean the pedestal and make a new blank measurement with a fresh aliquot of the appropriate buffer.
Hi Mohamed, I am using a protocol modified from QIAGEN AllPrep DNA/RNA Mini kit protocol as I need to co-extract both DNA and RNA at the same time. Have you ever work on stool b4?
Are you blanking with the exact same buffer bottle that you eluted with? We keep some extra kit buffers around for blanking on the nanodrop - but after a couple months, they have a tendency of evaporating, getting contaminated with dust, and giving wildly different concentrations than what you get if you use the exact same bottle to blank and elute. It might not be immediately noticeable in a sample with a high concentration, but in a low concentration it can give negative numbers.
Hi Yuan-Yeu Yau, I have tried to clean after first time I got the result, but is still the same.
Hi Alexandra, yes, I am using the same buffer, my senior did that together with me and we are using the same buffer but her samples seems to get a fantastic results.
Assuming your Nanodrop machine and measurement are fine, the kit "QIAGEN Allprep extraction" you used might not be optimized for 'stool' DNA/RNA isolation. Because Qiagen also sell other kits specified for 'stool' DNA/RNA isolation. See below:
1. QIAamp Fast DNA Stool Mini Kit: [https://www.qiagen.com/us/shop/sample-technologies/dna/genomic-dna/qiaamp-fast-dna-stool-mini-kit/#orderinginformation]
2. AllPrep PowerFecal DNA/RNA Kit. For simultaneous purification of genomic DNA and total RNA from stool samples. https://www.qiagen.com/us/shop/sample-technologies/dna/genomic-dna/allprep-powerfecal-dna-rna-kit/#orderinginformation
I've had more luck with random number generators than nanodrops. Is it essential that you determine the concentration? If so, I would try a cuvette-type spectrophotometer. Best of luck finding a solution.
Cuvette-type spectrophotometer can be used for DNA concentration measurement. However, I think that the most convenient thing to use NanodropTM for DNA measurement is that it needs only a very little amount of samples (1-2 uL). It is very useful for measuring those precious DNA samples with small amount.
The sample chamber inside a cuvette cell of a cuvette-type spectrophotometer is usually large, and need to be filled with much more DNA for proper measurement. One can always dilute the DNA sample for measurement, but it can be a problem if the initial concentration of the DNA sample is already low. Ultra-micro Quartz cuvette cells (to hold 1 uL, 5 uL, 10 uL,.....DNA) are available (see below link). However, it is very expensive. From the link provided above, for 5 uL cell, it costs ~$1200 US dollar.
http://www.thomassci.com/Laboratory-Supplies/UV-Vis-Chromatography-Cells/_/Ultra-micro-Cells?q=Small%20Volume%20Spectrophotometer
Hi Yuan-Yeu, I didn't check on a gel and not a cuvette-type spectrophotometer either. The kits (AllPrep PowerFecal DNA/RNA Kit) is just started to be available from QIAGEN this year as told by the customer care hotline staff just now when I called. What I suspect is the high fat concentration of my sample which hinder the extraction process, I have tried troubleshooting the protocol and will try it again tmr
Hi Clara Chong,
Make sure that your blank is clean but also make sure that there are no air bubbles when you pipette the sample to do the reading. Air bubbles can obstruct the light path of the spectrophotometer, which leads to inaccurate measurements. I had negative results once and that was the issue.
Hope this helps
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