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ZebraEz's Content

There have been 3 items by ZebraEz (Search limited from 16-July 18)


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#167084 Is that normal the negative control showing signals?

Posted by ZebraEz on 16 April 2014 - 01:20 PM in PCR, RT-PCR and Real-Time PCR

Have you had a look at the melt peak? It may be primer-dimer...

 

For the record, what are your Ct values for lowest concentration sample and NTC, respectively?

 

Note: If possible, could you post images of the amplification curves and melt peaks? It would make it a lot easier to understand the situation...




#167062 Is that normal the negative control showing signals?

Posted by ZebraEz on 15 April 2014 - 01:20 PM in PCR, RT-PCR and Real-Time PCR

I get that quite often, but it is just attributed to non-specific or background amplification. As long as the sample Ct is significantly lower that the NTC, then you can ignore this.




#167040 qPCR cannot detect reaction.

Posted by ZebraEz on 14 April 2014 - 04:04 PM in PCR, RT-PCR and Real-Time PCR

Hi.

 

I use a LightCycler 2.0 qPCR machine, with KAPA mastermix (SYBR Green).

 

I have encountered a problem regarding the machine detecting the samples (before the run actually starts).

 

To put it simply, the machine is failing to detect samples, with the sole exception of 18S.

 

It has only started with a fresh aliquot of mastermix (the older aliquot works fine), and for subsequent aliquots I have thawed to check for mastermix aliquot contamination.

 

The other primers I have tried are two different NANOG aliquots, and SOCS3, all of which have worked previously on older mastermix aliquots.

 

I have ruled out machine error, as I have been loading the capillaries in non-linear order, and then re-attempting with a shuffled order, with all samples in triplicate.

 

I have been using blank samples (NTCs) while I have been trying to figure this out.

 

Thanks,

Ezra.





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