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Old 05-25-2016, 10:45 AM   #1
ABABNEH
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Default Good results on qubit , but none in the bioanalzyer

Dears:
i am using the KAPA Stranded RNA-Seq
Library Preparation Kit to prepare my Libraries from RNA starting material. i had a very good success rate using this kit , but yesterday after the PCR amplification step and clean up step with the beads the qubit concentration of my libraries was in the range of 1-19 ng/ul. but in the bioanalyzer i had no peaks for my libraries . any suggestions? and this is the first time it happened with me ???
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File Type: pdf PEAK.pdf (23.6 KB, 52 views)
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Old 05-25-2016, 12:13 PM   #2
Ola
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The first sample has a very high adaptor dimer peak. Qubit only measures total DNA.
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Old 05-26-2016, 07:54 AM   #3
pmiguel
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Yes, the primer dimer peak is double-stranded DNA. So Qubit will give you its concentration. That peak is very, very high -- notice that it is 1500 fluorescence units in the chromatogram.

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Old 05-31-2016, 09:13 AM   #4
ABABNEH
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Many thanks for both of you, so any idea what got wrong ?
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Old 05-31-2016, 10:57 AM   #5
fanli
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Too little starting material so you ended up with mostly primer dimer. Was your input sample any different this time around?
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Old 05-31-2016, 11:26 AM   #6
pmiguel
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Yeah, forgetting to add the input DNA would be the simplest explanation. But anything that could go wrong but still allow adapter dimers to form, could be the culprit.

Your only strong clue is that the failure is catastrophic. That is, you didn't just come in at 50% of where you have in the past. Instead you are at least 100X low -- probably more.

So you are looking for something major going wrong.

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