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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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.
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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.
Thanx
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