Delivery of PE2 or PE2 variants into the cell library

MS Myungjae Song
JL Jung Min Lim
SM Seonwoo Min
JO Jeong-Seok Oh
DK Dong Young Kim
JW Jae-Sung Woo
HN Hiroshi Nishimasu
SC Sung-Rae Cho
SY Sungroh Yoon
HK Hyongbum Henry Kim
request Request a Protocol
ask Ask a question
Favorite

To deliver each PE2 variant to cell library A or B, PE2 variant-, pcDNA-BSD-, and puro-eGFP-encoding plasmids were mixed at a weight ratio of 10:1:1 to yield a total of 12 μg (for experiments using library 1) or 24 μg (for library B) of plasmid mixture, which was then transfected into a total of 1 × 106 cells from cell library A or a total of 6 × 106 cells from cell library B using Lipofectamine 2000 (Invitrogen), following the manufacturer’s protocol. After incubation overnight, the culture medium was exchanged with DMEM containing 10% FBS and 40 μg/ml blasticidin S (InvivoGen). Five days later, the transfected cells were harvested with 0.25% trypsin for genomic DNA extraction and deep sequencing.

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

post Post a Question
0 Q&A