Hi, I had prepared library for c. elegans small RNA sequencing using v1.5 small RNA illumina kit. I should got 93 and 100 bp bands in all the samples after PCR enrichment by agilent bioanalyzer. But I am getting only 100-102 bp in three and 106-109 in the remaining 5 samples. The primer dimer is 71 base pair length that i had cut out after running samples on 6% native DNA PAGE gel. If any body have any practical idea about this problem then plz let me know.
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Just to be sure I understand this:
After the PCR enrichment, you purify the construct (93 and 100 bp bands,) running the samples on a 6% native DNA PAGE gel (so the adapter-only construct is left apart). Then, you run the agilent bioanalyzer, where you get the 100-109 bp ? -
small RNA library preparation
Hi Cascoamarillo,
Thanks for your reply and you understand it correctly. Now what you suggest?Comment
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Ok. Looks like you have some stuff between the adapters, so I suggest to clone it and sanger sequence to see what size distribution you really have (one plate or half for 100-102 bp and another for 106-109). That's the only way to be sure before going to deep sequencing. It would be helpful to see the chip picture (HS DNA chip?). In my agilent chips, you see a peak around 100, but it's really hard to confirm the boundaries of the library; and when you sequence it, the size distribution can be slightly different from when it looked like in the chip.
BestComment
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miRNA library preparation
Hi everyone,
It is going to be my first next generation sequencing experience. I will use GS FLX for sequencing miRNAs. I would appreciate if anyone could tell me the pros and cons of the two library preparation methods, blunt end ligation and basic amplicon sequencing.
ThanxComment
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GS FLX is not recommended for miRNA sequencing due to their sizes. The protocols are optimized to size select much larger fragments not to mention short reads will be all trimmed during analyzis.Comment
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