Dear colleagues,
I would like to isolate whole bacteria (with more or less undamaged DNA) from plant roots. The roots are currently at -20°C. My goal is to disrupt plant tissue in a way that gains plenty bacterial cells, but at the same time does not damage the bacterial cells too much. I base my isolation on Ikeda et al. 2009. I don't have a lot of root material (something like 1 gram per sample...), and currently can't do an enzyme degradation of the tissue. Do you think it is better to grind the roots in a coffee grinder (where the sample gets heated), or macerate them with cooled steel mill (which could be rougher to the cells but will ensure a cooler temperature)?
Hi, I have no experience this and I would be potentially interested in the answers myself, just two notes from me:
First, I would say that freezing the roots may not be the best idea. But it's true that bacteria in general survive freezing, so hopefully it shouldn't matter.
Second, how about enzymatic dissolution of cell wall and separation of protoplasts (you can simply burst them by osmotic pressure) and the bacterial cells? That would appear like nice (albeit expensive probably) method to me.
Tomáš Hluska I looked into enzymatic digestion methods but for the first insight into my topic I have to use what my lab has in the storage, - sadly no appropriate enzymes. Therefore I am currently trying to optimize the physical plant tissue disruption and centrifugation procedure.
Also I thought about using the previously frozen samples... not perfect indeed. But my logic was, the roots also sometimes get frozen in the soil, so I hope the damage to the entire system won't affect the bacteria too much.
hi
you can run the direct PCR . you can test the protocol with non-original sample :
1- 1g root tissue wash with 200 ul TE buffer in 1.5ml micro tube by vortex (2-5 min ) Note : if bacteria is present on the outer surface of root tissue . if bacteria sample is " Rhizobium leguminosarum " , you should chop the "root nodules" by Scalpel in 500ul TE buffer.
2- 1ul of bacteria mixture in TE buffer used as template in 25 ul reaction PCR work . ( denaturation 94c 5 min , disrupt cell wall of bacteria and release DNA to mixture )
3- check PCR by "house-keeping" genes such as (16S, tus, rpoD, glyA, dnaB, gyrA, pykA/F, pfkA/B, mdoG, arcA)
4- if result is ok , you could save bacteria in TE buffer (-20 c ) for 1-2 year .
good luck
The classic method is grinding with mortar, pestle, some sea sand and a lot of elbow greese. You'll need some buffer because of the acidic content of the lysosomes, adjust the osmotic pressure with sucrose or some other polyol. Add only a little buffer initially and more as the mass becomes homogeneous.
you can just use a blender machine (like Ultra-Turrax T25 apparatus (IKA, Janke & Kunkel, Germany). Also grind and mortar is quite efficient. For protoplast depends of what you want but I follow this article (Birnbaum et al K, https://www.nature.com/articles/nmeth0805-615) concerning root protoplasts, it was helpful for me. Good luck
Yes, Grinding of dried roots using an ultra blender/grinder will be the best option to choose for your purpose.
You may use sonicator/ ultrasonic vibrator for this purpose.
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