Hi everyone,
I am in dire need of a method for DNA isolation from herbarium specimens (up to 5 years old or so) of woody plants (mainly Lauraceae, Moraceae and such). I have used some classical method (non-CTAB one) but without much success. I have also used Qiagen Kit, but as the yield is low and the plant material is low in quantity I need a better yield. I have looked into the literature, and I think best way could be the use of CTAB method with PVP. But if anyone has personal experience in such methods or can suggest me a proper logical way to standardize it, it'd be highly appreciated.
Please opine. Thanks.
CTAB method is one of the most reliable DNA extractions methods in plants. It has one problem - if tissue has a lot of polysaccharides, it is difficult to clean up DNA for some specific applications like GBS or PacBio sequencing. However, in your case I would do multipliable parallel extraction with Qiagen kit if DNA quality is satisfactory. This is time saving strategy.
I use the stratec kit for pinus RNA extraction and it works well. I suppose you can use their DNA kits as well.The only thing is polysaccharide conteining plants can work not that good..
Thank you everyone for replying.
Phani and Norbert, thanks for suggesting me alternatives for the usual procedures.
Zhebentyateva, incidentally I do have to subject these samples for Sanger (and maybe NextGen) sequencing, do you think if I use BSA while PCR and Exosap treatment before sequencing PCR that would help me cut out the contamination?
BSA will help and before sequencing you should anyway do a clean up of your PCR reaction. Every company has quite cheap kits for that or you precipitate your DNA again...
Thanks Norbert. For PCR clean up, I have kits, Exosap and PEG, so I think one of these should go well.
When I and my colleagues worked with herbarium specimens, we applied CTAB method but homogenized plant material adding a bit of glass powder. You can easily make this of cover glasses for microscopy. This improves mechanical degradation of hard material thus improving DNA extraction.
The Macherey-Nagel extraction kit can be also used for the nucleic acids isolation from woody plants - we have a broad experience in forest tree species population genetics and have tried many kits and methods. Sometimes, application of the direct-PCR largely help to overcome the difficult step of the DNA isolation.
Raunaq, BSA and Exosap treatment are used for completely different purposes - BSA usually helps to protect enzymes while digestion. ExoSap is used to remove primers and dNTPs from PCR mix before sequencing.
Keep to use Qiagen Kit and using the young tissue of your sample or multiplied
I am using the CTAB method with 0.01gm of PVP for 100mg leaf samples of Vitex glabrata. I found a good result. 5 micro L betamarkeptoethanol should be added after grinding tissues. You can also follow the Doyle and Doyle, 1987 protocol.
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