DNA extraction from endive (low yield + peak at 220 nm) - (Mar/11/2014 )
A few days ago I did a DNA extraction of leaf samples of endive using a protocol based on magnetic beads (unfortunately I cannot tell the name of the DNA isolation kit).
The yield was low and after I put my sample on the Nanodrop I saw a peak at 220 nm. According to literature it could be the presence of polyphenols. The next isolation I did was with PVPP (I couldn't find PVP on the lab) and with b-mercaptoethanol (in the lysis buffer). After DNA isolation I still saw the peak at 220 nm. I think that polyphenols are not the problem here.
What could it be? And how could I increase my yield? (i was thinking about ethanol or isopropanol precipitation).
PVPP is a highly cross-linked version of PVP, I'm don't think it will be effective in removing polyphenols as it isn't very soluble, but that's more of a guess than actual knowledge.
The gold standard for these sorts of extractions is to do a PVP and CTAB extraction (both are quite cheap), followed by phenol-chloroform-isoamyl alcohol extraction, which used to work fine for me many years ago (late 90's) on pittosporum samples, which are chock-full of polyphenolics and related compound.
PVP can be found in different molecular weights, if you have to buy, I would try to get the PVP 10000 rather than PVP 40000. It should give better results.
I used a protocol based on CTAB and PVP followed by the chloroform:IAA like bob1 but without phenol, to get high molecular weight DNA from some fungal isolates for genome sequencing and I got good quality DNA.
Reference: Sahu, Sunil Kumar, Muthusamy Thangaraj, and Kandasamy Kathiresan. 2012. “DNA Extraction Protocol for Plants with High Levels of Secondary Metabolites and Polysaccharides Without Using Liquid Nitrogen and Phenol.” ISRN Molecular Biology 2012: 1–6. doi:10.5402/2012/205049. http://www.hindawi.com/isrn/molecular.biology/2012/205049/.
I was already thinking about CTAB but I was curious what the 220 nm peak and the low yield could be. PVP 10000 is already ordered. @El Crazy Xabi: I will also take a closer look at the protocol suggested by you.
I just measured my CTAB elution buffer and there was no 220 nm peak. And the yield was 20 times higher than the other DNA isolation method. For further experiments CTAB will be used ;)