Melting Curve giving fluctuating Ct values

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Melting Curve giving fluctuating Ct values

Post by Newbie » Thu Jul 13, 2006 5:29 pm

Hi all,

I am doing some QPCR experiments with SYBR Green using cDNA and my primers never seem to work properly. I re-designed it 3 times already, the first time the primers looked promising- from the melting curve there were no primer dimers but then the CT values were flutuating up and down for my dilutions. So I re-designed the primers and by this I only mean that I only changed the amplicon regions and they were even worse because both gave me primer dimers. I really feel like the first primer set should work because there was amplification and no primer dimers but I just don't understand why the CT values don't behave! Anyone one have any idea??? I'm getting a little weary of re-designing the primers because it seems like I'm just randomly picking new primers and hoping they will work. But if I do have to re-design it again does anyone have any tips as to how to design the primers that may be more efficient? I follow general guidelines keeping the Tm at around 55-65, the GC content is ~50%, and avoiding more than 2 C and/or G's in the last 5 nucleotides. I also designed the primers pairs so that they each would lie on a different exon. Is there anything else I should consider? I heard it was better to have the primers end in C's or G's instead of A's or T's, is that true? Is it better if the primers span the exon borders or to lie on different exons? What else can I change? Ahhhhhhhh........

luna-celeste
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Post by luna-celeste » Thu Sep 07, 2006 3:59 pm

Maybe it's not a problem of primers but a problem of dilution.
At the beginning it's quite difficult to succeed in doing it.
Just try again with your first primers, you may need practice.

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