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Amplification curve in the negative control samples - (May/26/2014 )

I have a problem in my PCR. In the negatve control samples, I have a signals although my target is not in there. 

Can the SYBR green bind to genomic DNA ؟ Although I designed primers for a specific gene ( HA of H5N1) 

-Mohamed 1984-

You can blast the primers against the human genome. I assume the sample gDNA was isolated from a human sample. It's unlikely that both primers would target the same region in the human genome to allow for amplification of a product similar in size to your HA specific product. Did you validate your primers with end-point PCR - determine the annealing temperature or MgCl2 conc, and run the product on the gel? See if it is producing the single band free and free of primer dimers. It's most likely primer dimer. Happens in the negative control sometimes. Did you still have your PCR product from the negative control that produced a signal? You can run it out on a gel to determine if it is a primer dimer. I am guessing the Ct or Cq value is around 35-45 for this neg control? 

 

Hope this helps!

-Epigeneticist-

This is right, the CT values is around 35 or 34.

-Mohamed 1984-

Yes, SYBR can bind any double stranded DNA. Including primer dimer and non-specific products.

Also contamination.

 

Melting curve and/or running it on gel can say more about what it is.

If it's contamination you want to get rid of it, if it's nonspecific probably it would be present in the possitive samples as well. If it's a dimer (much lowr Tm) present only in negative control and not in your samples you don't need to worry about it.

-Trof-

Mohamed 1984

 

I forgot to include that point about the melt curve that Trof mentioned. I am not sure if you have included this step post amplification. If not, you need to include it for Sybr. Sometimes my negative control or negative samples produce a late signal but I can clearly tell it is a non-specific product by looking at the melt curve.

 

Also, keep track of the position of your neg ctl in your plate or other samples in general. Maybe you run your negative in the same well, such as a corner of the plate. I've had issue with the plate seal and samples near the perimeter of the plate gave weird signals. The sample produced amplification curves but they definitely did not look real. Strong signal then they just dropped off. Just something to consider for future runs.   

 

 

 

Yes, SYBR can bind any double stranded DNA. Including primer dimer and non-specific products.

Also contamination.

 

Melting curve and/or running it on gel can say more about what it is.

If it's contamination you want to get rid of it, if it's nonspecific probably it would be present in the possitive samples as well. If it's a dimer (much lowr Tm) present only in negative control and not in your samples you don't need to worry about it.

-Epigeneticist-

Thanks all. The point that I am amplifying HA gene of influenza meaning that it is strange to find it in the non infected animal ( the control negative animals). I got a signals in these samples in the brain and lung as well in all replicates. I did not make DNAse digestion during RNA isolation because qiagen kit elimenate all gDNA. However, if the cause is the gDNA, It is stilll very strange for me how to find the HA gene in the non infected samples ???? It gives a CT of 35 or more .

Any explanation

Thanks

-Mohamed 1984-

Thanks all. The point that I am amplifying HA gene of influenza meaning that it is strange to find it in the non infected animal ( the control negative animals). I got a signals in these samples in the brain and lung as well in all replicates. I did not make DNAse digestion during RNA isolation because qiagen kit elimenate all gDNA. However, if the cause is the gDNA, It is stilll very strange for me how to find the HA gene in the non infected samples ???? It gives a CT of 35 or more .

Any explanation

Thanks

 

 

There is where you need to figure out what you are amplifying - is it primer dimer, contamination or primer specificity issue with the gDNA. You cannot figure out what this product is unless you look at the melt curve and also run a gel. You need to figure out the product size and melt temperature. I usually do not trust anything with a Ct > or = 35. If you are consistently amplifying this product and it is not primer dimer, then you can sequence and blast it. 

 

You can use UCSC In-Silico PCR to see if these primers are hitting anything other than your HA target but I really think it is primer dimer or low level contamination (i.e. carry over). 

http://genome.ucsc.edu/cgi-bin/hgPcr?command=start

-Epigeneticist-

Hi All, 

Although I am appreciating every of these comments. I do not think it is a contamination issue since I changed the place and all equipment. I thought that it might be a problem of excess primer concentration so I diluted the primer 10 Pm, 7 Pm, 5 Pm and 2,5 Pm then I tried all dilutions with NTC and + ve samples. All the dilutions gives signals in both but I found a 10 CTs lower in the sample than in the NTC. attached are the values . I think I trust now the idea that if 5-10 ct higher in the NTC, this indicate backgroung signals 

 

Any comments

 

 


Attached Image

-Mohamed 1984-