View Single Post
Old 12-20-2018, 11:59 PM   #3
Kujin Kwon
Junior Member
 
Location: Korea, South

Join Date: Dec 2018
Posts: 7
Default

Thanks luc.
I adopted protocol from here.
https://onlinelibrary.wiley.com/doi/...755-0998.12529

However, it doesn't fit well with our lab's reagents so I changed some conditions.
This protocol is for Truseq but I need iSeq library. Because of this reason, after reverse transcription, I designed primer which can attach both 3',5' region of cDNA and have a sequence for i7 and i5 adapter site.

The reason why I set annealing temperature as 72C is dimer problem. Actually, I started PCR from 65C but lots of dimer found. Also, I found dimer melting temperature peak at 70C in qPCR output.
When I changed melting temperature at 72C, total amount of sample was increased by 3~4 fold (7 cycle)

You can check some images that I uploaded
1. Gel pic before gel elution.
2. qPCR after 72C results
Attached Images
File Type: jpg 2018_12_21_010147_(Nucleic Acid)_raw.jpg (43.0 KB, 9 views)
File Type: png qPCR_after_72C.PNG (117.5 KB, 6 views)
Kujin Kwon is offline   Reply With Quote