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expession only in +ve controls


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4 replies to this topic

#1 George250

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Posted 14 April 2009 - 06:44 AM

Hi

I have strong bands in my positve controls but no bands in my samples. What that means? What goes wrong? (expression of the gene should occur in these samples tho...).

Thank u

#2 almost a doctor

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Posted 14 April 2009 - 07:01 AM

Hi

I have strong bands in my positve controls but no bands in my samples. What that means? What goes wrong? (expression of the gene should occur in these samples tho...).

Thank u



Hi George, we need loads more information to try and help you, as from your post we really have no idea what is that you are doing.
My guess is that you are talking PCR, but just because you posted on Mol Bio forum, and you are talking about bands...

Tell us more details and we'll see what we can do.

Edited by almost a doctor, 14 April 2009 - 07:01 AM.


#3 George250

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Posted 14 April 2009 - 07:13 AM

Hi

I have strong bands in my positve controls but no bands in my samples. What that means? What goes wrong? (expression of the gene should occur in these samples tho...).

Thank u



Hi George, we need loads more information to try and help you, as from your post we really have no idea what is that you are doing.
My guess is that you are talking PCR, but just because you posted on Mol Bio forum, and you are talking about bands...

Tell us more details and we'll see what we can do.


I am realy sorry for this..

Well I am doing conventional PCR which a gene of interest.. This gene should be expressed in all samples, although I had expression of this gene only in my potive controls.. There was no expression in the rest of the samples.. Also my RT and RNA -ve controls appeared blank at the gel..

Hope this helps! Thank you very much!!

#4 almost a doctor

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Posted 14 April 2009 - 07:20 AM

Hi

I have strong bands in my positve controls but no bands in my samples. What that means? What goes wrong? (expression of the gene should occur in these samples tho...).

Thank u



Hi George, we need loads more information to try and help you, as from your post we really have no idea what is that you are doing.
My guess is that you are talking PCR, but just because you posted on Mol Bio forum, and you are talking about bands...

Tell us more details and we'll see what we can do.


I am realy sorry for this..

Well I am doing conventional PCR which a gene of interest.. This gene should be expressed in all samples, although I had expression of this gene only in my potive controls.. There was no expression in the rest of the samples.. Also my RT and RNA -ve controls appeared blank at the gel..

Hope this helps! Thank you very much!!


Hey, still need more information, but here's what I can think of with what I have, your problem is on your sample.

What's your positive control? What's your sample? You say that RT and RNA -ve controls are blank, which I'm assuming is RT reaction without enzyme and RNA (non-RT), so you are doing RNA extraction and then RT-PCR. Are you sure your RNA is fine? Are you sure your RT works?

Really need more information. If you controls worked the problem is not on the PCR (reaction and reagents are working), so you have to track down where the problem might be.

Cheers

#5 George250

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Posted 14 April 2009 - 07:35 AM

Hi

I have strong bands in my positve controls but no bands in my samples. What that means? What goes wrong? (expression of the gene should occur in these samples tho...).

Thank u



Hi George, we need loads more information to try and help you, as from your post we really have no idea what is that you are doing.
My guess is that you are talking PCR, but just because you posted on Mol Bio forum, and you are talking about bands...

Tell us more details and we'll see what we can do.


I am realy sorry for this..

Well I am doing conventional PCR which a gene of interest.. This gene should be expressed in all samples, although I had expression of this gene only in my potive controls.. There was no expression in the rest of the samples.. Also my RT and RNA -ve controls appeared blank at the gel..

Hope this helps! Thank you very much!!


Hey, still need more information, but here's what I can think of with what I have, your problem is on your sample.

What's your positive control? What's your sample? You say that RT and RNA -ve controls are blank, which I'm assuming is RT reaction without enzyme and RNA (non-RT), so you are doing RNA extraction and then RT-PCR. Are you sure your RNA is fine? Are you sure your RT works?

Really need more information. If you controls worked the problem is not on the PCR (reaction and reagents are working), so you have to track down where the problem might be.

Cheers



My positive control is mouse placentae and my samples embyos.. I am sure that my RNA is fine because I have used the same RNA to test b-actin and I had expression in all samples (positive plus samples)... If u need for info let me know!! thanx a lot!!!!!!!!!!!!!!!!




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