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...
________________________________ From: "[email protected]" <[email protected]> To: [email protected] Sent: Sun, June 26, 2011 7:04:04 PM Subject: Methods Digest, Vol 73, Issue 11 Send Methods mailing list submissions to [email protected] To subscribe or unsubscribe via the World Wide Web, visit http://www.bio.net/biomail/listinfo/methods or, via email, send a message with subject or body 'help' to [email protected] You can reach the person managing the list at [email protected] When replying, please edit your Subject line so it is more specific than "Re: Contents of Methods digest..." 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 <[email protected]> Subject: RE: mammalian stable cell-line To: <[email protected]>, methods_magpie <[email protected]> Message-ID: <[email protected]> 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: [email protected] > Date: Sat, 25 Jun 2011 04:28:36 +0000 > To: [email protected] > Subject: Re: mammalian stable cell-line > > In article <[email protected]>, >=?iso-8859-1?B?UGVwYSBGbG9yZXogUOlyZXo=?= <[email protected]> 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 > [email protected] > http://www.bio.net/biomail/listinfo/methods ------------------------------ Message: 2 Date: Sat, 25 Jun 2011 22:11:23 +0000 From: "Phelan, Paul J." <[email protected]> Subject: RE: mammalian stable cell-line To: Pepa Florez P?rez <[email protected]>, "[email protected]" <[email protected]>, methods_magpie <[email protected]> Message-ID: <4b4b5298fa967b4d90d7ed983b9214cb085df...@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: [email protected] [[email protected]] on behalf of Pepa Florez Pérez [[email protected]] Sent: Saturday, June 25, 2011 2:17 PM To: [email protected]; 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: [email protected] > Date: Sat, 25 Jun 2011 04:28:36 +0000 > To: [email protected] > Subject: Re: mammalian stable cell-line > > In article <[email protected]>, >=?iso-8859-1?B?UGVwYSBGbG9yZXogUOlyZXo=?= <[email protected]> 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 > [email protected] > http://www.bio.net/biomail/listinfo/methods _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods ------------------------------ _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods End of Methods Digest, Vol 73, Issue 11 *************************************** _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
