13 replies to this topic

[Mafalda]

Dear BioForum Members,

For weeks now, we are trying to isolation genomic DNA from Bacteria spores without much success. We used various procedures (sonication, beat beating, heating, microwaving, bleach treatment) and we also tried different combinations of the listed procedures in order to open the exosporium and get access to the DNA. We also used different extraction methods (special kits for gram positive bacteria and simple phenol-extraction protocols) to clean the DNA. However, non of the numerous attempts were fruitful and resulted in mensurable amounts of gDNA We have used spore concentration that range from approximately 1 x 10^5 to 50 x 10^6 spores per sample. At this point we are not sure what we could improve in order to get gDNA - with other words we are not sure if we get DNA out of the spores and lose it later or if we are not even able to break open the spores. Is there anybody who can help us since we have run out of ideas?!

[pito]

Mafalda, on Feb 8 2010, 09:53 AM, said:

Dear BioForum Members,

For weeks now, we are trying to isolation genomic DNA from Bacteria spores without much success. We used various procedures (sonication, beat beating, heating, microwaving, bleach treatment) and we also tried different combinations of the listed procedures in order to open the exosporium and get access to the DNA. We also used different extraction methods (special kits for gram positive bacteria and simple phenol-extraction protocols) to clean the DNA. However, non of the numerous attempts were fruitful and resulted in mensurable amounts of gDNA We have used spore concentration that range from approximately 1 x 10^5 to 50 x 10^6 spores per sample. At this point we are not sure what we could improve in order to get gDNA - with other words we are not sure if we get DNA out of the spores and lose it later or if we are not even able to break open the spores. Is there anybody who can help us since we have run out of ideas?!

It might help if you tell us what protocol you use, how you work.
It is possible you make some mistake during the proces that is causing you to loose the dna, but now we dont know how you work.

[gebirgsziege]

what makes you think you have not enough gDNA? i.e. Is your PCR not working, do you see insufficient bands on a gel, have you been doing a picogreen measurement....if you could share some more details somebody might be able to help you.

[Mafalda]

gebirgsziege,

"what makes you think you have not enough gDNA? i.e. Is your PCR not working, do you see insufficient bands on a gel, have you been doing a picogreen measurement....if you could share some more details somebody might be able to help you."

>> Well, pcr is working but there are no bands on the gel. We have used something similar to picogreen measurement and found very low concentrations. We need larger amounts of gDNA in order to clone it.

pito,

"It might help if you tell us what protocol you use, how you work.
It is possible you make some mistake during the proces that is causing you to loose the dna, but now we dont know how you work."

>> We tried a lot of different protocols that should help to open the spores. The problem is that there is an infinite number of combinations and adjustments (beat beating for 5", 10", 15",...1' or whatever) we could try. It seems that there is not a general way to open spores but maybe there is a way to test if any of the methods is suitable to open the spores we are working with.
>> We were wondering if people working with bacterial spores have made similar experiences. Is it possible that any of the numerous proteins associated with the spore surface could bind or degrade the DNA? We have used MasterPure™ Gram Positive DNA Purification Kit, QuickExtract™ Bacterial DNA Extraction Kit, and Phenol/chloroform protocols. We could isolated DNA in the presence of other bacteria or DNA to test such possible interference. Any other suggestions?

[gebirgsziege]

As you have been trying such a huge amount of different extraction protocols, you might have a PCR problem?

So have you quantified the DNA content in you gDNA or only the PCR product?

You are using such a huge amount of spores, probably you need to dilute your DNA (inhibition of the PCR by too much DNA). Try 1:10, 1:100, 1:1000, 1:10000 dilutions of your sample, probably you will be suprised.

Are you sure your primers work correctly and you PCR conditions fit your primers?

Do you have an option of using nested PCR prior cloning?

[Mafalda]

gebirgsziege, on Feb 9 2010, 11:49 AM, said:

As you have been trying such a huge amount of different extraction protocols, you might have a PCR problem?

>> The DNA isolation produces only very low concentration of gDNA that are not visible on the gel. However, using the extracted DNA as PCR template works perfectly fine - we are getting strong single bands! For the PCR we only need one copy but we intend to use the gDNA for cloning and therefore we need higher recovery rates.

[gebirgsziege]

think finally I got you

Why don't you simply try to extract more spores?

Or an alternative which works very fine is a Whole Genome Amplification prior to cloning (for example see here).

Edited by gebirgsziege, 09 February 2010 - 03:46 AM.

[Mafalda]

gebirgsziege, on Feb 9 2010, 12:45 PM, said:

Why don't you simply try to extract more spores?

It is not so easy to get spores - and we are already using large amounts of it. I think if we would improve the extraction we might increase the amount without using more spores.

gebirgsziege, on Feb 9 2010, 12:45 PM, said:

Or an alternative which works very fine is a Whole Genome Amplification prior to cloning (for example see here).

Whole Genome Amplification is not a option - we have tried it and the results were very disappointing ranging from genome bias to chimera products, and primer extension problems. We would prefer clean gDNA.

Thanks for your suggestions!

[gebirgsziege]

sounds like a hard one then; but one more try: what if you resuspend your pellet in less water/TE at the last step of DNA extraction?

[HomeBrew]

When I used to work in Bacillus (anthracis, thuringiensis, subtilis, etc.), we used to do a modified version of Kado and Liu (J. Bacteriol. 145:1365-1373) as is described here. It was quite effective.

May I ask why you don't grow the spores and extract the DNA from vegetative cells? Also, you mention that "it is not so easy to get spores" -- can't you just prepare more from vegetative cells by allowing them to sporulate?

[Mafalda]

HomeBrew, on Feb 10 2010, 12:33 AM, said:

When I used to work in Bacillus (anthracis, thuringiensis, subtilis, etc.), we used to do a modified version of Kado and Liu (J. Bacteriol. 145:1365-1373) as is described here. It was quite effective.

May I ask why you don't grow the spores and extract the DNA from vegetative cells? Also, you mention that "it is not so easy to get spores" -- can't you just prepare more from vegetative cells by allowing them to sporulate?

> The bacteria we are working with is an obligate parasite. We collect spores because earlier stages are difficult to harvest. The spores can be washed and isolated easier from possible (host) contaminants.

> Thanks for the citations! - In fact , we used a similar phenol-chloroform method and that's why we think the problem lies in an earlier step i.e. the opening of the spores.

[HomeBrew]

Mafalda, on Feb 10 2010, 06:25 AM, said:

The bacteria we are working with is an obligate parasite. We collect spores because earlier stages are difficult to harvest. The spores can be washed and isolated easier from possible (host) contaminants.

Aha -- I asked this question gently because I knew there had to be a valid reason you were dealing with spores and that they were hard to get...

Mafalda, on Feb 10 2010, 06:25 AM, said:

Thanks for the citations! - In fact , we used a similar phenol-chloroform method and that's why we think the problem lies in an earlier step i.e. the opening of the spores.

Prior to the phenol:chloroform step, the 60C incubation in SDS/NaOH/sucrose/Tris followed by digestion with pronase (or proteinase K) was critical. If you use proteinase K, that digestion can be performed at 60C also. You might need to jack up the heat and or time of incubation in the lysis step prior to pronase/proteinase K digestion -- as I recall, some people would even use an autoclave.

[Mafalda]

HomeBrew, on Feb 10 2010, 02:33 PM, said:

Prior to the phenol:chloroform step, the 60C incubation in SDS/NaOH/sucrose/Tris followed by digestion with pronase (or proteinase K) was critical. If you use proteinase K, that digestion can be performed at 60C also. You might need to jack up the heat and or time of incubation in the lysis step prior to pronase/proteinase K digestion -- as I recall, some people would even use an autoclave.

> Thanks I will give it a try! Is there a possibility to check if there is DNA after the incubation prior to the phenol:chloroform step?

[HomeBrew]

I suppose you could try running a bit of the lysate on an agarose gel and staining it with etidium bromide -- I'm not sure what you'll see, though.