I'm trying to extract DNA:
The extraction buffer consists of Tris-HCl, NaCl, EDTA and SDS. I put 600ul of the extraction buffer for extraction using steel beads and homogenizer, followed by addition of 20ul of proteinase k, incubate at 65°C for 2 hours. Then centrifuge to obtain the supernatant. Add equal volume of isopropanol to the supernatant, invert and rest at room temperature 5 min, then centrifuge to discard the supernatant. The dna pellet is a bit yellowish or brownish color. The color maintained even though i washed it with 70% ethanol twice and finally elute with 20ul of preheated TE buffer.
Even after elution, it is yellowish and it seems like it is not fully dissolved in the buffer even after i heat it at 50°C for 10 min. When i try to quantify it using a uv spec, it was around 2500ng/ul (30mg of leave sample) and 7000ng/ul (60mg leave sample) (Previously i used kit to extract and almost never get more than 300ng/ul, so i'm a bit skeptical)
Q1) Is it normal to get yellowish DNA pellet? or is the yellowish thing the protein?Will UV spec give reading if its protein?
Q2) Why cant the DNA pellet fully dissolved?
Q3) Will the yellowish thing inhibit downstream analysis like PCR, cloning, etc?
Q4) How to get rid of the yellowish color?
Any opinions/suggestions are welcomed and much appreciated.Thank you.
DNA is color less and 2500 ng/ul is redeciosly high. Your DNA is probebly contaminated by some polyphenols such as tannins or lignin.
Hi Vikki,
Are you trying to extract plasmid DNA? If so then thousands of ng/ul does sound high. The concentrations that you're giving make it sound like you've got bacterial genomic DNA or something included. I typically use kits for my DNA extractions but the advice that I can give is from when I do protein extractions from E. coli. I follow similar steps to you (lysis etc) and typically the supernatant that I get at the end is slightly yellow. I always just thought it was something in the E. coli that gave it a slight yellow tint. This almost always disappears when I use a column for purification (whether it's a miniprep column or a Ni-NTA spin column etc).
I've done plenty of gigapreps with DNA concentrations going up to 25mg/ml and never had a problem with oddly coloured DNA. If your DNA is contaminated with protein you could certainly see that on UV spec as you would have peaks at 280nm and 230nm but I'm fairly sure that the isopropanol and 70% ethanol you've added would get rid of contaminating protein and I don't know why it would be yellow.
Could you potentially try re-extracting your DNA with a column? And have you tried running your DNA out on an agarose gel to see what it looks like?
Mark
Try ceramic beads. Steel attrits to produce steel fines that can vary in color from yellow to brown. These are small enough to become suspended by brownian motion and would come out during centrifugation.
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