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Old 01-03-2014, 06:52 PM   #1
jiangyue0203
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Question Unexpected signal in 700-1000bp region by Agilent Bioanalyzer

Hi guys!

I keep having a problem with the final RNA-seq liabrary size. After enrich DNA fragments, everything seems to be OK on the electrophoretogram with DNAmaker I (see attached thumbnails) and we chose gel-method to obtain the fragments (300-400bp) and purified using MinElute Gel Extraction Kit (QIAGEN). BTW, we eluted the liabrary by 25ul RNA-free water instead of QIAGEN buffer EB.

Then we used Agilent Bioanalyzer to obtain the exact fragments size and found every sample had a unexpected signal between 700-1000bp region!(see attached thumbnails)

Does anyone have this problem before? Do you have any idea what can cause this unexpected signal? I swear no irrelevant fragments were extracted during my experiments.

Many thanks!!!
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Old 01-09-2014, 01:04 PM   #2
newmoon
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it could be ssDNA if you do to many cycles of PCR
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Old 01-22-2014, 05:51 AM   #3
james hadfield
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You've over amplified the library with too many PCR cycles and are seeing a "bubble' product. This is where the PCR reaction runs out of primer but annealing means the ends of 2 non-complementary inserts anneal at P5/P7 creating a bubble fo single stranded DNA in the middle. This run differently on the Bioananlsyer

You can QT with qPCR and seqeunce as normal but might want to adjust your PCR cycles down.
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