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Old 05-04-2010, 10:07 AM   #1
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Location: New orleans

Join Date: Feb 2010
Posts: 3
Default insert size

I am doing RNA-seq using illumina kit. But i am confusing on the insert size. When performing gel seclection after cDNA being ligated with adaptor, the 200 bp size with cDNA and adaptor is seclected on gel. Because the adaptor length on the two end of cDNA is 60 bp, the cDNA should be 140 bp. After performing PCR using 200 bp size (adaptor plus cDNA) as template, the final product should be 260 bp. Because the each PCR primer has 30 nt overhang compared with template, in theory, the final size should be 200 + 60 (sense and antisense primer), 260 bp. However, the factual size is still around 200 bp by Agilent bioanalyzer from illumina protocol. How to explain the difference between theoretical 260 bp and realistic 200 bp? What's wrong with the agilent analysis or gel seclection?
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