It's hard to tell what the problem is without more details, but most of my sequencing failures and frustrations have been due to gel purifications that are not pure enough for the sequencer. After lots of trial and error, I have found that 3 - 4 minutes in a speedvac after purification will get rid of residual ethanol, but I haven't found anything that can remove traces of agarose or guanidium salts (binding buffer), which will definitely result in sequencing failure. I check quality with a Nanodrop and only sequence samples that have a 260/230 of 1.5 or better.
On Sun, Jun 26, 2011 at 1:37 PM, Nikola Loncar <[email protected]> wrote: > 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 > _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
