We are examining the potential for DNA damage in individual shoot tips following various treatments in a plant conservation program. We already know of the various kits available commercially and also have seen some published protocols, but these typically require 5g of tissue (which we do not have). We are also dealing with plant tissue, rather than animal tissue, which may introduce additional difficulties. We would be very interested in hearing from others who may have dealt with this problem before. Thanks.
We usually use 100mg of plant tissue and we have very high and good quality of DNA. Using an modificated CTAB protocol (Doyle & Doyle, 1987) AND we usually start the extraction macerating the tissue with liquid nitrogen, then start the CTAB protocol.
In fact we use the liquid nitrogen with kits (Invitrogen Plant Kit), using like CTAB protocol, 100mg of the sample.
EDITED:
> We're working with pines, oaks, sycamore, oregano, and other plants.
Please contact the resource person in the following
www.helinibiomolecules.com
You will get solution .......
Find the article in the link provided. They have mentioned about large scale (6g) and small scale (1g) isolation protocols.
http://www.nature.com/nprot/journal/v1/n6/abs/nprot.2006.471.html
Isolation of Arabidopsis nuclei and measurement of gene transcription rates using nuclear run-on assays.
Kevin M Folta1 & Lon S Kaufman2
Try Qiagen DNA isolation kit or Manual CTAB protocol.
As per my experience, you try CTAB protocol. We also regularly use CTAB protocol with 30mg plant sample for thousands of samples.
I will suggest that you try the Qiagen plant DNA extraction kit. It is efficient for such sample size as you mentioned. Thanks
You can try the protocol in the link
https://www.researchgate.net/publication/261713631_High_Quality_Genomic_DNA_impurities-Free_from_Sugar_Crops_and_Other_Plant_Tissues?ev=prf_pub
I tried it more times
Conference Paper High Quality Genomic DNA impurities-Free from Sugar Crops an...
Thanks everyone for your comments. We have already tried the standard CTAB extraction protocol and our available tissue (just a few mg) appears to be too low for the Qiagen kit, but we will try the FTA card method and see how it goes. Out main problem is that we are limited to very small amounts of tissue (just an individual root tip for an individual) that have been cryopreserved for several years.
How much tissue do you have ? DNA extraction from 100mg is a routine. If you send me an e-mail. [email protected]
You can try the direct PCR kits with some modification. Small amount of fresh sample is used for direct PCR and the results are sustainable.
I use 30mg plant tissue for DNA isolation using CTAB method. Since the amount of tissue is small, crush it in ependroff tube. Rest all protocol will be same.
you can use the Automatic extractor MagMax Magnetic Particle procesor from Applied Biosystems. It is excelente for very small quantity!
My suggestion that you should try different type methods for genomic DNA isolation.
You can use this protocol, it is very useful
Conference Paper High Quality Genomic DNA impurities-Free from Sugar Crops an...
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