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No amplification in qPCR, but band in standard PCR

No amplification in qPCR, but band in standard PCR - Real-Time PCR and Quantitative PCR Forum

No amplification in qPCR, but band in standard PCR - Real-Time PCR and Quantitative PCR Forum


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Old 09-24-2013, 11:04 AM
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Default No amplification in qPCR, but band in standard PCR



I'm having a bit of a strange problem with qPCR. I have been trying to replicate an experiment from a paper, using their primers and conditions. One of the primer sets work but the control never amplifies. I repeated the PCR exactly with the same polymerase and conditions etc, using standard PCR, and got a band on the gel. So the PCR does work, but won't work in qPCR. Any suggestions? Has anyone else encountered this? I'm using SYBR green.
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Old 09-25-2013, 12:55 PM
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Default Re: No amplification in qPCR, but band in standard PCR

how big the amplicon is, SYBR usually works on amplicon of 60-150bp, it won't work if if it is over 200bp
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Old 02-15-2014, 11:51 AM
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Default Re: No amplification in qPCR, but band in standard PCR

qPCR mixes contain additives that lower annealing temperature. You have to go down by 5C compared to the optimum for ordinary PCR.
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amplification , band , pcr , qpcr , standard


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