problem with 16S rRNA sequncing

Cathal Garvey via methods%40net.bio.net (by cathalgarvey from gmail.com)
Mon Jun 27 04:08:43 EST 2011


If agarose is the problem, try using agarase?

My money is on the gel extraction though. Stop using UV and see how you get
on; brief UV illumination reduces transformation frequencies with DNA
tenfold, I imagine it does sequencing no favours either. Try crystal violet,
or methylene blue for easy post-electrophoresis staining methods. If you
have patience, order sybr-safe, use a blue LED to excite and filter with
orange Perspex. If you want a custom-tailored solution, the guys at
pearlbiotech.com make a lovely integrated gelbox specifically designed to
facilitate real-time observation of a sybr-safe gel running.

Of course, I have repeatedly heard on this list that the best form of gel
extraction is by centrifuging liquid (&DNA) from a gel fragment by
filtration. Fragments are placed on a bed of (cellulose? Acetate?) in a
0.5ml tube with a hole punched in the bottom, placed into a 1.5ml tube, and
spun. DNA is then precipitated from the recovered liquid. Possibly you're
supposed to freeze the gel first, not sure.

Finally, you could avoid the issue if you can get your gel extracted
fragment to amplify in another round of PCR. If you can get a PCR reaction
to deliver just one band, you should be able to send it off after a quick
precipitation or cleanup, I'd expect.
On 27 Jun 2011 04:33, "Sandy Emme" <sandysu57 from gmail.com> wrote:
> 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 <hemicar085 from yahoo.com>
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: "methods-request from oat.bio.indiana.edu"
>> <methods-request from oat.bio.indiana.edu>
>> To: methods from magpie.bio.indiana.edu
>> 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
>> Methods from net.bio.net
>> http://www.bio.net/biomail/listinfo/methods
>>
>>
>>
>> ------------------------------
>>
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>> Methods mailing list
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>> http://www.bio.net/biomail/listinfo/methods
>>
>> End of Methods Digest, Vol 73, Issue 11
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