DNA extraction from dry cells - How to extract DNA from dry cells (Oct/06/2008 )
I have a trouble with DNA extraction from dry cells
My sample is a fouling membranes, onwhich there is some bacteria communities. I want to search for the diversity of bacteria on those membranes
Unfortunatetly, my samples (fouling membranes) are brough from UK, and they were dried as you dry a A4 paper.
I have to extract genomic DNA from those membrane and use for further study
I use sonication to disrupt the cells (the membrane is put into the buffer over night, because i want the cell absorp some humidity), and use for ultrasonication for 4 times (20-30 sec/treatment)
Phenol/chloroform/isoamyl (25:24:1) was then used to purify DNA
Alcohol was used to precipitate the DNA,
DNA is diluted in to TE buffer (small volume)
But...I don't have final band on electrophoresis 
Does any one have experience with DNA extraction from dry cells????Please give me some tips.
Appriciate all your help
Happy
I have never extracted DNA from dried sample, but have you tried a digest with 10ug/ml pronase with 1% SDS in TE? Incubate for at least 2hrs at 55 Celsius.
You could also hydrate your sample for 10 minutes in TE and SDS, that freeze it. Later thaw the sample at 55C and add pronase.
You could also come along after that with a cell lysis protocol using a gunidium thiocynate solution. Followed by a phenol purification step.
You could also hydrate your sample for 10 minutes in TE and SDS, that freeze it. Later thaw the sample at 55C and add pronase.
You could also come along after that with a cell lysis protocol using a gunidium thiocynate solution. Followed by a phenol purification step.
Im also thinking of the problem is mainly in cell disruption step. All the cells are attached on membrane which is dried.
I found that, the cells can not be detach well from mebrane by using sonication/ultrasonication. I am planning to try with bead-beating.
Does any one have ideals about DNA from dried cells? (dried bacteria). Do you think the DNA still there?
Do you think the some of DNA could be degraded in dried cells???
It depends how fast the sample was dried and how much moisture remained in the sample. If drying was slow, there is more time for the sample to degrade. If the sample has high moisture content when 'dried', the DNA sample would also degrade.
As I understand, if done correctly, drying a sample can preserve a DNA sample. Although freeze drying or immersion in ethanol are more preferred methods for preserving DNA in the field.
If you have a large sample to work with I guess there would be DNA. Bacteria cells are more numerous compared to a sample from animal tissue. Some DNA should be extractable, unless the preservation process when very very wrong.
Alcohol was used to precipitate the DNA,
DNA is diluted in to TE buffer (small volume)
stupid question (and probably insulting), did you add salt (sodium acetate) to the dna before adding the alcohol?
When the drying is done properly, there should be no problem with your DNA besides you are working with highly fragile bacteria???
So I think the cell rupture is the "problem" in your preperation. Maybe your bacteria encapsulated when they were dried and your cell rupture with sonication is not sufficent to get them open? I would try to rinse the Bacteria form the membrane with some mild detergent (Tween 80 0.5% or SDS/CTAB buffer) and then use a bead beater if you can AND add the membrane with the attached bacteria in a bead beater.
And btw have you tried to PCR your DNA extract? Maybe I misunderstood your post, but are you mainly concerned because you cannot see any DNA in your extract? Because PCR can work perfect even if you could not see any DNA on a gel before 
DNA is quite strong, I have purified DNA (ok it is plasmid), put it on a piece of paper dried it and send it to another lab so they would have the construct.
I have done it myself dissolved that piece of paper with the dna and electropored it into e-coli and multiplied it.
In my experience, if you do not see it on gel, it does not mean that it is not present.
Agarose gel is not so sensitive. Just do a PCR on it and try a fiew times, it might still be there but in to low amounts for gel analyses.
So like earlier sugested by the others it will probably be your cell disruption itself.
i've used the Nucleon Phytopure kit to extract DNA from herbarium specimens of algae that are several decades old, some over a hundred years old! it hasn't always worked but many times it does.
Thanks for your all comments and suggestions
My sample is the fouling membrane, which is dried previously by other group working on desalination I've check how people dried them...and It seems, drying was not correctly as we normally treat our sample (frozen or using liquid N2...)
I've tried following method
1, I thought mainly the reason of failure in my extraction is lied on Detaching the biofiml on membrane and cell disruption step. Therefore Instead of using ultrasonication, i tried with bead-beating:
-cut the membrane in small piece, put in to buffer (Tris_HCl 50mM, EDTA 10mM and NaCL 1.5M + RNAase)
-store over night
-and add the glass bead
-followed by further voltex and
-using phenol. chloroform, isoamyl alcohol purification,
- then ethanol precipitation
I found that all the bio-film was detached from membrane, and alot of pellet i got at the end. I did tried to qualify DNA sample by nano-drop and electrophoresis. No band at all on gel (6uml sample/well) even the nano-drop show DNA concentration about 20-100 ng/uml. (A260/280 ~1.5-1.7)
I did tried PCR, in which the template was 100 ng for 50uml reaction, 35 cycles...but it was failed, no band on the gel at the end
2, today i tried again by using kit (MOBIO, Soil kit).
Using the blade to scale the biofilm on membrane (which is dumped in to buffer overnight). Then centrifuge to collect all the cell pellet. Using that sample for DNA extraction using kit....
God, I failed again, no band on gel...I am planning to check PCR, amplifing 16S rDNA as some of you recommended...but i wonder of failure.
I did PCR using Kit also (premix PCR kit). That kit was use successfully in all every PCR exp before. Therefore there is no reason for PCR reaction or technique...Also the final DNA product was dissolved into Tris-HCl buffer, and use immidiately for PCR (not using TE, so no inhibition of EDTA for PCR)
Question:
1, Do u think the dried samples have no DNA orgirinally
2, Do u think the cell disruption step have problem?
3, What do u think the number i got from nano-drop, it was quite high, isn't it? but what is it? Plasmid?/ RNA?
4, Other recommendation???? Please! 
Yeah, I did Dear!
1/10 Vol. of sodium acetate + 2 Vol. of 100% ethanol ---> on -20 for >4 hours
Cleanup by ethanol 70%
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I am considerring alot about your suggestion. I am thinking of using probe ( Using DAPI for FISH) to see is there still DNA on it???? How do u think about this idea?
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Well, It is probably...
I've have experience of extracting DNA from some forrest trees. it's so hard at that time also, even the leaves were fresh. I grinded the sample in liquid N2 and used this mind detergent which had SDS, CTAB and PVP (Polyvinyl Pyrrolidone). High conc of NaCl was used/ i got the success after 2 months for developing the protocol
Do you think this detergent (CTAB, PVP) is neccessary for my buffer, to disrupt the cells which may encapsulated????