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3 replies to this topic

#1 vetticus3

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Posted 28 November 2013 - 09:09 AM

Hi,

has any one had a problem where there are certain samples that reach the maximum threshold at the first cycle (ie, there is no amplification curve just a flatline at max levels).... but there is a product that is melting at the correct temperature?

To make matters a bit stranger... the samples which are looking "correct" have a melting curve that is about 10'C coller than normal... and are have a much later than normal ct?

 

This is a machine or software problem?

I have changed

primers

water

samples

re run samples that have previously worked

 

it is the same position for the max amplifications.... but still having everything else melting at a cooler temp... i have never had this happened before and it's driving me mad.

 

thanks,

v



#2 phage434

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Posted 28 November 2013 - 04:11 PM

The dye is sensitive to dsDNA. Perhaps you are adding far far too much template DNA, such that the dye binds and becomes fluorescent with no amplification whatsoever. Cycling could still produce a band at the correct size.



#3 vetticus3

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Posted 29 November 2013 - 01:58 AM

Ah, but that the rub.  I've used these sample before, and had no problem with it.  Also, I found out last night, that if I use the same standard curve (which produces that flatline) on a different position, it turns to rubbish - way too much variability between samples.

Still, I've got a shift of 10'C, a melt curve that looks like bad modern art, and previously verified samples turning bad.

 

Has anyone had problems with software corruption?  or filters getting dirty?  Either it's me (not the samples... but actually me doing bad voodoo on the experiment) or it's the machine/software.



#4 vetticus3

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Posted 02 December 2013 - 07:17 AM

in case anyone has this issue... it's the gain function on the program.

check with the user manual on how to adjust this.






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