qPCR help - have you ever seen amplification plots like these?

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teaelleceecee
teaelleceecee's picture
qPCR help - have you ever seen amplification plots like these?

I'm trying to perform a real time PCR with bulk mosquito samples, but every time I try, I get weird amplification plots (view: http://i.imgur.com/MqLqXN9.jpg). When I perform the PCR without SYBR, it works fine (I get amplicon bands on a gel), when I perform it with SYBR, I get no amplicon bands on a gel. It's a ~350bp fragment (incl. primers tailed with Illumina adaptors) and the sample is DNA extracted from 100 mosquitoes which have been homogenised (16 samples in total).

The first plot is the DNA just on its own in the reaction. The second is the DNA serially diluted 1:2, until the lowest concentration is 4 ng/ul. The third plot is trying siluted DNA with a different SYBR (the first 2 PCRs were done with 1.5ul of x100 SYBR added with MyFi Taq + Buffer, and primers; the third PCR was done with a SYBR master mix and primers).

I've never amplification curves like this before - I originally thought I had way too much DNA or SYBR, which is why I tried dilutions and a different SYBR, but neither seemed to have worked.

Has anyone seen amplification plots like this before? And if so, what's causing this issue/how can I fix it?

Brad Best
Brad Best's picture
Well, you've kind of answered

Well, you've kind of answered your own question.  The amplification plots look like no amplification at all, and you did the gel to prove it.  For some reason, your assay doesn't play nice with either the SYBR or the cycling conditions you're using for qPCR.

Are you doing PCR and qPCR under identical conditions, other than presence/absence of SYBR?

teaelleceecee
teaelleceecee's picture
Yes, I did the PCR and qPCR

Yes, I did the PCR and qPCR under the exact same cycling conditions. I don't know what in my sample would not work with SYBR....