problem with 16S rRNA sequncing

[Nikola Loncar]

Hi,

I have a problem with 16S rRNA sequncing. I am trying to use this to identify 
bacterial strain isolated from ground. I am using 20F and 1492R primers and I am 
getting PCR product at annealing temp at 45 C. I also used one atcc strain in 
order to compare sequences and to have control of my experiments. I did PCR with 
pfu, gel extraction, gel analysis of extracted fragments (only single bands, no 
contamination present) but when I send these for seq i receive nothing. first 
they said that it is possible to have more than one strain in wt isolates, but 
when i told them that i have atcc among samples they told me to do everyhting 
again, so i did. same result.. allegedly, this is something that happens 
sometimes when someone tries to identify bacillus strains by this method, but 
now i suppose to find solution by myself.. if anyone had this kind of problem 
before and came up with solution please respond...  





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Sent: Sun, June 26, 2011 7:04:04 PM
Subject: Methods Digest, Vol 73, Issue 11

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Today's Topics:

   1. RE: mammalian stable cell-line (Pepa Florez P?rez)
   2. RE: mammalian stable cell-line (Phelan, Paul J.)


----------------------------------------------------------------------

Message: 1
Date: Sat, 25 Jun 2011 20:17:59 +0200
From: Pepa Florez P?rez <snipeurope from hotmail.com>
Subject: RE: mammalian stable cell-line
To: <dk from no.email.thankstospam.net>, methods_magpie
    <methods from magpie.bio.indiana.edu>
Message-ID: <BAY166-w5889BD4DFF7B70126F182CD3550 from phx.gbl>
Content-Type: text/plain; charset="iso-8859-1"


First of all, thank you very much for all your answers.

Maybe I haven't explained it properly. but what we are interested in ordering is 
a mammalian stable-cell line (Hek-293 or Hela S3), that stable expresses a 
recombinant protein.
Is it possible to get that from ATCC?

Thank you!!


> From: dk from no.email.thankstospam.net
> Date: Sat, 25 Jun 2011 04:28:36 +0000
> To: methods from magpie.bio.indiana.edu
> Subject: Re: mammalian stable cell-line
> 
> In article <mailman.122.1308950757.14889.methods from net.bio.net>, 
>=?iso-8859-1?B?UGVwYSBGbG9yZXogUOlyZXo=?= <snipeurope from hotmail.com> wrote:
> >
> >Hi=2C
> >
> >we are thinking about ordering a mammalian stable cell-line=2C probably usi=
> >ng HeLa S3 or Hek-293 cells.
> >
> >Could anyone recommend me some companies and give me any information about =
> >which prices are reasonable?
> 
> ATCC is a gold standard for common cell lines. Anything else is not going 
> to be much cheaper to justify a small risk of getting a not-quite-the-same
> cell line.  
> 
> DK
> _______________________________________________
> Methods mailing list
> Methods from net.bio.net
> http://www.bio.net/biomail/listinfo/methods
                          

------------------------------

Message: 2
Date: Sat, 25 Jun 2011 22:11:23 +0000
From: "Phelan, Paul J." <Paul.Phelan from tufts.edu>
Subject: RE: mammalian stable cell-line
To: Pepa Florez P?rez <snipeurope from hotmail.com>,
    "dk from no.email.thankstospam.net" <dk from no.email.thankstospam.net>,
    methods_magpie <methods from magpie.bio.indiana.edu>
Message-ID:
    
<4B4B5298FA967B4D90D7ED983B9214CB085DF42B from TFTMEXDAG01MB03.tufts.ad.tufts.edu>
    
Content-Type: text/plain; charset="iso-8859-1"

Yes, go to www.atcc.org and do a product search for the cells you want, they 
should have them.

________________________________________
From: methods-bounces from oat.bio.indiana.edu [methods-bounces from oat.bio.indiana.edu] 
on behalf of Pepa Florez Pérez [snipeurope from hotmail.com]
Sent: Saturday, June 25, 2011 2:17 PM
To: dk from no.email.thankstospam.net; methods_magpie
Subject: RE: mammalian stable cell-line

First of all, thank you very much for all your answers.

Maybe I haven't explained it properly. but what we are interested in ordering is 
a mammalian stable-cell line (Hek-293 or Hela S3), that stable expresses a 
recombinant protein.
Is it possible to get that from ATCC?

Thank you!!


> From: dk from no.email.thankstospam.net
> Date: Sat, 25 Jun 2011 04:28:36 +0000
> To: methods from magpie.bio.indiana.edu
> Subject: Re: mammalian stable cell-line
>
> In article <mailman.122.1308950757.14889.methods from net.bio.net>, 
>=?iso-8859-1?B?UGVwYSBGbG9yZXogUOlyZXo=?= <snipeurope from hotmail.com> wrote:
> >
> >Hi=2C
> >
> >we are thinking about ordering a mammalian stable cell-line=2C probably usi=
> >ng HeLa S3 or Hek-293 cells.
> >
> >Could anyone recommend me some companies and give me any information about =
> >which prices are reasonable?
>
> ATCC is a gold standard for common cell lines. Anything else is not going
> to be much cheaper to justify a small risk of getting a not-quite-the-same
> cell line.
>
> DK
> _______________________________________________
> Methods mailing list
> Methods from net.bio.net
> http://www.bio.net/biomail/listinfo/methods
                                          
_______________________________________________
Methods mailing list
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http://www.bio.net/biomail/listinfo/methods



------------------------------

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