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Multiple Bands in qPCR

Primer multiple Bands qPCR

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4 replies to this topic

#1 AlexanderA

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Posted 21 December 2012 - 03:14 AM

Hello there,

I am facing a problem with a new Primer set I designed and I hope that someone can help me with this.

I designed primers for SYBR Green qPCR and ran a PCR with temperature gradient from 57-62°C.
The Primers are blasted and are specific for my sequence. One splice variant is also amplified which has the same amplicon size.
The no template control and no rt control did not show any amplification.
I see a broad peak in the melt curve of my product at many temperatures. At some temperatures the meltcurve looks good to me. I ran the products on a 2% Agarose Gel.
Multiple Bands larger than my specific product (120bp) can be seen on the Gel.

Is there anybody who can help me out with this?

Best regards Alex

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#2 Trof

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Posted 21 December 2012 - 04:26 AM

Since your unspecific bands are bigger than your product, you can try decreasing the elongation time. Or annealing if it won't help, since the temperature gradient didn't help.
Thought I would say 58 °C looks pretty fine.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

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#3 AlexanderA

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Posted 21 December 2012 - 05:42 AM

Hi Trof,

thank you for your quick reply. I think I will try to decrease elongation time.
But just fort the understanding: What could the larger products be? The gDNA Amplicon should be approximately 16kb, which is quite unlikely to be amplified and obviously not on the gel.

Best regards Alex

#4 Trof

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Posted 21 December 2012 - 06:54 AM

Hard to say, it can still be nonspecifities. Or transcripts not published/bad spliced.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#5 AlexanderA

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Posted 10 January 2013 - 12:56 AM

Hi,

I just wanted to add a comment about my results. I did the qPCR again on another Cycler with rapid cycling (decreased annealing and elongation time), another mastermix, and annealing at 58°C.
The melting curve looks great now, everything is fine Posted Image

Best regards, and thanks again

Edited by AlexanderA, 10 January 2013 - 12:56 AM.






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