Methods Digest, Vol 36, Issue 6

[Jim Young]

I have done similar knock-down. I am sure that bad transfection will cause a
lot of problems. Why do you use si RNA oligonucleotide instead of vectors.
You can have a siRNA expression vector to express siRNA for you. The results
are much better.  You can see the links for details:
http://www.genscript.com/targeting_vector_construction.html
http://www.genscript.com/siRNA_service.html

On Thu, May 8, 2008 at 1:06 PM, <methods-request from oat.bio.indiana.edu> wrote:

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> Today's Topics:
>
>   1. Question (juan pablo morera rojas)
>   2. Luciferase Assay! (Sudheendra Rao N R)
>   3. Re: EmulsionPCR (Sudheendra Rao N R)
>   4. Re: co-transfecting with multiple vectors (Sudheendra Rao N R)
>   5. Re: co-transfecting with multiple vectors (Sudheendra Rao N R)
>   6. Re: Does Lipofectamine cause gene knock down? (Sudheendra Rao N R)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Wed, 7 May 2008 20:24:27 -0600
> From: "juan pablo morera rojas" <morera98 from gmail.com>
> Subject: Question
> To: "Huang Ke-xue" <methods from magpie.bio.indiana.edu>
> Message-ID:
>        <887f1c940805071924p7fc7d27dn5ef4575755522b41 from mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> Hi, my name is Juan Pablo Morera, Im from Costa Rica. Im sorry if Im
> bothering you in any way. I wanted to ask you about one primer Im working
> with and if you could help me. It has 52bp but I dont know why is so long,
> I
> found another paper that includes a shorter primer but still I have
> the longer one (it works by the way) besides it includes a enzyme
> restriction site, just adding an "A" in the end of the primer, but
> originally in the sequence there isnt site for any enzyme. If you have some
> clue about it I would really appreciate it.
>
> PSD I just know that it works but I dont know how does it.
> Thank you very much for your time and Attention.
>
> Juan Pablo Morera
>
>
> ------------------------------
>
> Message: 2
> Date: Thu, 8 May 2008 20:01:10 +0530
> From: "Sudheendra Rao N R" <sudhee26 from gmail.com>
> Subject: Luciferase Assay!
> To: methods <Methods from magpie.bio.indiana.edu>,
>        methods from magpie.bio.indiana.edu
> Message-ID:
>        <a1a1abbb0805080731o1841b171xd20d37076f51b153 from mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> Hi all,
> 1. I am doing Luciferase assay to understand transcriptional regulation. I
> was looking for proper reporter lysis buffers.
> i found out one with KH2PO4 (7.8 pH) with 0.2% Triton X-100 (v/v)..is there
> any better?
> it takes time for us to order promega 5x reporter lysis buffer.
>
> 2. i am doing duplicate assays in 6 well plates..and collecting cells in
> pbs
> and lysing them in 200uL buffer. however i am not sure what is the
> "standard" lysate:luciferase substrate (roche) proportion.
> i have tried 10uL:90uL, 40uL:60uL, 100:100uL combinations, later gives me
> better RLUs
> we use a stand-alone, tube-luminometer (sirius), and do not use injectors.
>
> please let me know.
> (i will post my questions on DLuciferase Assay also..soon :))
>
> thanks in advance
> sudheendra.
>
> --
> Think before agree
> Think before you nod
> but STOP thinking
> and You Are God
>
>
> ------------------------------
>
> Message: 3
> Date: Thu, 8 May 2008 20:13:51 +0530
> From: "Sudheendra Rao N R" <sudhee26 from gmail.com>
> Subject: Re: EmulsionPCR
> To: peter <peter.ianakiev from gmail.com>, methods
>        <Methods from magpie.bio.indiana.edu>
> Message-ID:
>        <a1a1abbb0805080743t43814238ub8619f7dc99873a from mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> i thought,
> vortexing itself would make the emulsion uniform
>
> On Tue, May 6, 2008 at 6:22 PM, peter <peter.ianakiev from gmail.com> wrote:
>
> > On May 2, 4:31 pm, peter <peter.ianak... from gmail.com> wrote:
> > > Hi group, I need to make emulsion in the emulsionPCR uniform. Any
> > > suggestions?
> > > Thanks,
> > > Peter
> >
> > What is going on? No experts in emulsion PCR on this board? No one
> > reads it?
> > Anyway good luck with your research
> > _______________________________________________
> > Methods mailing list
> > Methods from net.bio.net
> > http://www.bio.net/biomail/listinfo/methods
> >
>
>
>
> --
> Think before agree
> Think before you nod
> but STOP thinking
> and You Are God
>
>
> ------------------------------
>
> Message: 4
> Date: Thu, 8 May 2008 20:19:05 +0530
> From: "Sudheendra Rao N R" <sudhee26 from gmail.com>
> Subject: Re: co-transfecting with multiple vectors
> To: "Wendy Grus" <wgrus from umich.edu>, methods
>        <Methods from magpie.bio.indiana.edu>,
> methods from magpie.bio.indiana.edu
> Message-ID:
>        <a1a1abbb0805080749n527b264m1f12ca28f00fa2a9 from mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> Hey, look in to some of these
> http://www.springerlink.com/content/u3531825942t18v5/
>
> http://www.bio.net/hypermail/methds-reagnts/2001-September/090500.html
>
> http://www.bio.net/hypermail/methds-reagnts/2001-September/090499.html
>
> best,
> sudheendra.
>
> On Mon, May 5, 2008 at 11:49 PM, Wendy Grus <wgrus from umich.edu> wrote:
>
> > Hi all-
> > I am transfecting some HEK 293 T cells to transiently express a few
> > different genes.  Some of these genes are on pcDNA3.1 vectors while some
> > are
> > on pCI vectors.  Will having them on different vectors affect their
> > expression levels?  Will I have to play with the concentrations of each?
> >  Should I put all the genes in one vector (singly)?
> >
> > Thanks,
> > Wendy Grus
> > _______________________________________________
> > Methods mailing list
> > Methods from net.bio.net
> > http://www.bio.net/biomail/listinfo/methods
> >
>
>
>
> --
> Think before agree
> Think before you nod
> but STOP thinking
> and You Are God
>
>
> ------------------------------
>
> Message: 5
> Date: Thu, 8 May 2008 20:23:55 +0530
> From: "Sudheendra Rao N R" <sudhee26 from gmail.com>
> Subject: Re: co-transfecting with multiple vectors
> To: "Wendy Grus" <wgrus from umich.edu>, methods
>        <Methods from magpie.bio.indiana.edu>,
> methods from magpie.bio.indiana.edu
> Message-ID:
>        <a1a1abbb0805080753n7c2ee025kb363e81520a72d0a from mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> and this!
> http://www.protocol-online.org/biology-forums/posts/25380.html
> i have done triple transient transfections..but yes..cell death is little
> more than single/double. But i seed more cells than i usually for
> single/double..and it seems to work..
> plasmid expression can be altered depending on promoter
> sequence..especially
> multiple plasmid with different promoters..i try keeping them constant
> (CMV-all CMV, that way)
>
> best,
> sudheendra.
> On Thu, May 8, 2008 at 8:19 PM, Sudheendra Rao N R <sudhee26 from gmail.com>
> wrote:
>
> > Hey, look in to some of these
> > http://www.springerlink.com/content/u3531825942t18v5/
> >
> > http://www.bio.net/hypermail/methds-reagnts/2001-September/090500.html
> >
> > http://www.bio.net/hypermail/methds-reagnts/2001-September/090499.html
> >
> > best,
> > sudheendra.
> >
> >   On Mon, May 5, 2008 at 11:49 PM, Wendy Grus <wgrus from umich.edu> wrote:
> >
> > > Hi all-
> > > I am transfecting some HEK 293 T cells to transiently express a few
> > > different genes.  Some of these genes are on pcDNA3.1 vectors while
> some
> > > are
> > > on pCI vectors.  Will having them on different vectors affect their
> > > expression levels?  Will I have to play with the concentrations of
> each?
> > >  Should I put all the genes in one vector (singly)?
> > >
> > > Thanks,
> > > Wendy Grus
> > > _______________________________________________
> > > Methods mailing list
> > > Methods from net.bio.net
> > > http://www.bio.net/biomail/listinfo/methods
> > >
> >
> >
> >
> > --
> > Think before agree
> > Think before you nod
> > but STOP thinking
> > and You Are God
>
>
>
>
> --
> Think before agree
> Think before you nod
> but STOP thinking
> and You Are God
>
>
> ------------------------------
>
> Message: 6
> Date: Thu, 8 May 2008 20:44:05 +0530
> From: "Sudheendra Rao N R" <sudhee26 from gmail.com>
> Subject: Re: Does Lipofectamine cause gene knock down?
> To: "Kamalian, Laleh" <L.Kamalian from liverpool.ac.uk>,     methods
>        <Methods from magpie.bio.indiana.edu>, methods from magpie.bio.indiana.edu
> Message-ID:
>        <a1a1abbb0805080814j66f07772nbdd511af02aa6992 from mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> Hi,
> quite scary i must say :)
> but if i am getting u right, u are trying to knock-down a gene using siRNA
> /
> mock siRNA in a stably transfected cell line(parental), which is also ur
> control cell line (which u are treating with only lipofectamine)
>
> knockdown is more in control cell line!! than proper siRNA
> actually knockdown is present in all transfected cell lines!!
>
> your control cell line no longer a control cell line.
> any contamination in lifofectamine? or optiMEM?
> try chaning the tranfection agent..
>
>
>
>
> On Thu, Apr 24, 2008 at 2:43 PM, Kamalian, Laleh <
> L.Kamalian from liverpool.ac.uk>
> wrote:
>
> > Hi everyone
> > I have come across this problem.  I have stably transfected my cells
> using
> > Lipofectamine 2000 as the transfection reagent.  So far, I have been
> using
> > my parental cells as the calibrator during transient transfection.  Now
> for
> > the stable transfection I have used scrambled si RNA oligonucleotide to
> > transfect my cells and used a pool of these cells as my control.  Using
> > quantitative PCR to measure the mRNA of the colony cells and the control
> > cells,  my colony cells show about 70% gene knock down comparing to the
> > scrambled si RNA control cells, which is good.  However comparing to the
> > parental cells, the control shows about 90% less expression of the gene
> of
> > interest.  Once before during my transient transfection I had the same
> > problem when used "no DNA" as my negative control i.e. Lipofectamine
> without
> > any DNA.  At that time I had used western blot to measure the protein
> > instead of QPCR.  I had noticed that my negative control lysate had
> > considerable decrease in protein expression.  My question now is can
> > Lipofectamine or transfection condition cause the gene knock down?  It
> > especially doesn't make sense now that my scrambled RNA control cells
> have
> > been growing in the selective medial for at least 4 weeks now.  I am
> > thinking that even if the transfection condition had affected the cells
> > transiently, it can't effect the mRNA level after being eliminated from
> the
> > growth media.  Am I right or am I missing something in here?
> > Laleh.
> > _______________________________________________
> > Methods mailing list
> > Methods from net.bio.net
> > http://www.bio.net/biomail/listinfo/methods
> >
>
>
>
> --
> Think before agree
> Think before you nod
> but STOP thinking
> and You Are God
>
>
> ------------------------------
>
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