Wierd amplification pattern. - (Jun/30/2006 )

So some of my rxns are running fine...
but

I have some runs that curve to negative flouresence then swing back positive at log phase. I don't know what would cause negative detection like this.

any thoughts.

sybr green, pcr, 45 cycles.

Wow! It's the first time I see something like that! It looks really weird!

tell me about it.

The odd this is, the C(t) values are fairly accurate. And the replicates do the same thing....it might be something in my DNA, not sure.....


i mixed well, no bubbles.....


any ideas?

Unfortunately I have no idea what the problem could be. I tried to find out something on the AB website but I could only find that you can have negative picks when you have a too strong signal because the machine cannot read it and therefore it will interpret it as negative. But this doesn't explain why it then becomes positive...

oh

and the Tm values are spot on.....


so i believe the results, i'm just gonna guess there something wierd in the detection.

I too am having weird amp. profiles. They are not as sigmoidal as they used to be. Could it be that Sybr is old/contaminated?
I have attached a spreadsheet showing several examples...any insight is greatly appreciated.

sneth, I was unable to upload your image...but, 45 CYCLES??????

why so many? and at which cycle did the fluorescence appear? most results over 30, all results over 35, are quite possibly artifacts....


medhan, could you post a pic of how they looked initially? based on my experience, I do not see any particular problem that jumped out at me...but change over time is not good. have you had your bulbs replaced in your machine? and, how old is your stuff?

Thanks for the reply...the curves have definitely changed as you can see in the file attached. We are having a PM on the icycler next week, so I will ask the tech what he thinks. Any more thoughts, would be great!

yes 45 cycles. It's supposed to be an exhaustive amplification of 10ng <

I think i discovered the culprit.....maybe. The TE used to elute the DNA.

TE (10:1)



I am thinking the TE was actually made (1:10). This would cause an abundance of EDTA. The EDTA would probably chelate some of the mg ions needed for PCR. Some other tests with just the dna had a negative amplification effect. I know the EDTA would kill the PCR, i wonder if it would do something to the SYBR as well?

this image is here if you can't see it for some reason : http://www.sneth.com/images/rtpcr.jpg