Tweet
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?
-
Proteins are often described as the workhorses of the cell, and identifying their sequences is key to understanding their role in biological processes and disease. Currently, the most common technique used to determine protein sequences is mass spectrometry. While still a valuable tool, mass spectrometry faces several limitations and requires a highly experienced scientist familiar with the equipment to operate it. Additionally, other proteomic methods, like affinity assays, are constrained...04-04-2024, 04:25 PM -
Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...03-22-2024, 06:39 AM