7 replies to this topic

[Maverix]

Hey all, does anyone know te best way to extract Plasmid DNA from mycobacteria. Also a colleague called it episomal plasmid. What does episomal plasmid mean?
I was told you could use the mini prep kit for E.coli but just add lysozyme and glycine to weaken the cell wall. Has anyone used this before? Is there any other way of extraction?

Also most importantly, I have a Plasmid that is integrated inside Mycobacterium tuberculosis H37Rv. Would anyone know the best way to extract integrated plasmids?

Thanks a lot,
Maverix

[fishdoc]

Maverix, on Jul 22 2009, 09:07 AM, said:

Hey all, does anyone know te best way to extract Plasmid DNA from mycobacteria. Also a colleague called it episomal plasmid. What does episomal plasmid mean?
I was told you could use the mini prep kit for E.coli but just add lysozyme and glycine to weaken the cell wall. Has anyone used this before? Is there any other way of extraction?

Also most importantly, I have a Plasmid that is integrated inside Mycobacterium tuberculosis H37Rv. Would anyone know the best way to extract integrated plasmids?

Thanks a lot,
Maverix

Episomal would mean a piece of DNA that can exist by itself or integrated in the genome.

Never tried doing preps on Mycobacterium.

As for extracting the integrated plasmid, perhaps a restriction digest of the genome, make a library, and probe the library for the insert containing the plasmid? PCR the plasmid out? Does the plasmid integrate and excise under differing conditions? If so, do the prep under the conditions in which it has excised.

[T C]

Hey,

I doubt if it would be that easy.  The Cell wall is just too rigid...I guess you need to use a bead beater.  BTW there is a book on mycobacterial protocols, if you can get hold of it:

http://www.springerp...1385/0896034712

Best,
TC  

Maverix, on Jul 22 2009, 07:37 PM, said:

I was told you could use the mini prep kit for E.coli but just add lysozyme and glycine to weaken the cell wall. Has anyone used this before? Is there any other way of extraction?

[Maverix]

Hey Fishdoc, Thanks for your reply.

So if someone calls it an episomal Plasmids, means it is could be extracellular or integrated or either?

Also I have done restriction digestion before but what do you mean by make a library. I never got that concept.  And for probing, I dont have the plasmid map as the origin was unknown (I do have a sequence of the plasmid and may try it on Vector NTI) but do you have any other suggestions as to how I could get the in silica copy of the plasmid structure?
Also how do you know what probe to use. If I have the sequence, order the primers, will it make i easy to PCR the plasmid?
I dont think the plasmid integrates or excises under diff. cnditions, but will have to look into it. The thing is there is not much info available on the plasmid.

Thanks a lot! N

fishdoc, on Jul 22 2009, 07:18 PM, said:

Maverix, on Jul 22 2009, 09:07 AM, said:

Hey all, does anyone know te best way to extract Plasmid DNA from mycobacteria. Also a colleague called it episomal plasmid. What does episomal plasmid mean?


I was told you could use the mini prep kit for E.coli but just add lysozyme and glycine to weaken the cell wall. Has anyone used this before? Is there any other way of extraction?

Also most importantly, I have a Plasmid that is integrated inside Mycobacterium tuberculosis H37Rv. Would anyone know the best way to extract integrated plasmids?

Thanks a lot,
Maverix

Episomal would mean a piece of DNA that can exist by itself or integrated in the genome.

Never tried doing preps on Mycobacterium.

As for extracting the integrated plasmid, perhaps a restriction digest of the genome, make a library, and probe the library for the insert containing the plasmid? PCR the plasmid out? Does the plasmid integrate and excise under differing conditions? If so, do the prep under the conditions in which it has excised.

[Maverix]

Hey Veteran, Thanks for your reply! That books looks like a good idea!!

T C, on Jul 23 2009, 01:21 AM, said:

Hey,

I doubt if it would be that easy.  The Cell wall is just too rigid...I guess you need to use a bead beater.  BTW there is a book on mycobacterial protocols, if you can get hold of it:

http://www.springerp...1385/0896034712

Best,
TC  

Maverix, on Jul 22 2009, 07:37 PM, said:

I was told you could use the mini prep kit for E.coli but just add lysozyme and glycine to weaken the cell wall. Has anyone used this before? Is there any other way of extraction?

[fishdoc]

Maverix, on Jul 23 2009, 08:28 AM, said:

Hey Fishdoc, Thanks for your reply.

So if someone calls it an episomal Plasmids, means it is could be extracellular or integrated or either?

Also I have done restriction digestion before but what do you mean by make a library. I never got that concept.  And for probing, I dont have the plasmid map as the origin was unknown (I do have a sequence of the plasmid and may try it on Vector NTI) but do you have any other suggestions as to how I could get the in silica copy of the plasmid structure?
Also how do you know what probe to use. If I have the sequence, order the primers, will it make i easy to PCR the plasmid?
I dont think the plasmid integrates or excises under diff. cnditions, but will have to look into it. The thing is there is not much info available on the plasmid.

Thanks a lot! N

To your first question, my guess would be yes, that's what they'd mean.

When you make a library, you digest the genome, take that digest and clone the pieces into a vector. All those pieces of genomic DNA go into the vector, so you get a bunch of bacteria that each carry a plasmid with a different part of the genome in the plasmid.

If you have the sequence of the plasmid, that's all you need. You can design PCR primers to make probes and identify any colonies from your library that carry the integrated plasmid.

Amplifying the plasmid by PCR won't necessarily be easy even with the sequence. It will depend on how big the plasmid is, what the surrounding DNA is like (GC content), etc.

As for protocols to do all this, I don't have that for you. Just suggestions as to what MIGHT works. Others that have done similar work may have better suggestions.

[T C]

Hey,

I found the book.  But I cannot attach it in the PM, send me yr mail id and I can send it across.

Best,
TC  

Maverix, on Jul 23 2009, 08:00 PM, said:

Hey Veteran, Thanks for your reply! That books looks like a good idea!!

[123blockhead]

how about best way to extract genomic DNA from Mycobacteria?
Would appreciate if anybody can provide this info.
Thanks in advance