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plz guide me.i got many band in one well in agrose gel electrophoresis - PCR (Nov/02/2007 )

Hi,
i did real time pcr by using mesenchymal stem cells.i used VEGEF and Flt primers but i got many band in one well.i didn't undertand reason why i got this very strange type of result.how i improve it?plz anybody who know the reason of it plz guide me.i think may be it is due to contamination which occur during cDNA synthesis or during RNA isolation process.plz reply me

-malaika-

Did u perform normal PCR and see ur desired band before going for real time ???????

-newarray-

You must have only one band when using real time RT-PCR, so it is convenient for you to check before.
Did you optimize your PCR reaction before going to PCR? There are several ways you can try to eliminate the spurious bands: optimize your annealing temperature and Mg2+ concentration, or maybe you're using too much primers or template, which can also decrease the specificity of the reaction. Did you check your primers efficiency?
Also you may be simply amplifying different splice variants. Are there any described for your gene? If that is the case, you will have to change your primers set, in order to avoid that region.

-SLAR-

QUOTE (SLAR @ Nov 2 2007, 10:04 AM)
You must have only one band when using real time RT-PCR, so it is convenient for you to check before.
Did you optimize your PCR reaction before going to PCR? There are several ways you can try to eliminate the spurious bands: optimize your annealing temperature and Mg2+ concentration, or maybe you're using too much primers or template, which can also decrease the specificity of the reaction. Did you check your primers efficiency?
Also you may be simply amplifying different splice variants. Are there any described for your gene? If that is the case, you will have to change your primers set, in order to avoid that region.


Thanks for your reply.yes i have optimize my pcr reaction i check differeny cytokines and their receptor and growth factors expression not only VEGEF and FLT.i got positive result for HGF and others.how i can optimize the mg ion concentration because we use kit method (kit from Qiagen).Annealing temperature of VEGF and Flt was 51.65 i run it 52 than 53 but i got same result there was no chnage.
plz tell me now what can i do for getting good result.

-malaika-

QUOTE (newarray @ Nov 2 2007, 09:23 AM)
Did u perform normal PCR and see ur desired band before going for real time ???????

No i didn't perform normal PCR before performing realtime PCR.

-malaika-

Hi,

usually every Taq comes with a vial of MgCl2 for optimization, even when you don't need it. That is why you can almost always decrease the concentration of MgCl2 to a minimum (usually PCR buffers have MgCl2 added, others don't). However, if you have unwanted fragments, you should be decreasing, not increasing your MgCl2 concentration. Therefore, I think you should better optimize your reaction by normal PCR in terms of annealing temperature (make a gradient in your thermal cycler and use other temperatures; probably there will be one that works), primer concentration (strongly recomend you to check primer eficiency), template concentration. If desperate measures are necessary try additives (like DMSO and others) and chelating agents (e.g. EDTA, to try to reduce MgCl2 concentration a little bit, if nothing else works).

Good luck!

-SLAR-

QUOTE (SLAR @ Nov 5 2007, 08:10 AM)
Hi,

usually every Taq comes with a vial of MgCl2 for optimization, even when you don't need it. That is why you can almost always decrease the concentration of MgCl2 to a minimum (usually PCR buffers have MgCl2 added, others don't). However, if you have unwanted fragments, you should be decreasing, not increasing your MgCl2 concentration. Therefore, I think you should better optimize your reaction by normal PCR in terms of annealing temperature (make a gradient in your thermal cycler and use other temperatures; probably there will be one that works), primer concentration (strongly recomend you to check primer eficiency), template concentration. If desperate measures are necessary try additives (like DMSO and others) and chelating agents (e.g. EDTA, to try to reduce MgCl2 concentration a little bit, if nothing else works).

Good luck!

Hi,
thanks for your kind consideration.i got too many band in reverse tranciptase PCR too.i think it is due to primers.
how can i check my primer efficiency?

-malaika-

Hi,

usually Real time PCR machines have that option. Usually you make severall dilutions of your sample DNA and the software will draw a graph which gives you (by the slope) the efficiency of your primers. What usually happens is that your primers are working but you're using too much sample so, you get unexpected bands. Check your software for this option, it should explain what you should do! If it doen't try to find it in other company pages (ex. BioRad, Roche, etc.).
Hope it helps.

-SLAR-

QUOTE (SLAR @ Nov 7 2007, 02:33 AM)
Hi,

usually Real time PCR machines have that option. Usually you make severall dilutions of your sample DNA and the software will draw a graph which gives you (by the slope) the efficiency of your primers. What usually happens is that your primers are working but you're using too much sample so, you get unexpected bands. Check your software for this option, it should explain what you should do! If it doen't try to find it in other company pages (ex. BioRad, Roche, etc.).
Hope it helps.

Thanks.but i wants to know if the same problem occue in reverse tranciptase PCR thgen what should i do?

-malaika-