Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/04/23

From OpenWetWare
Jump to navigationJump to search
Neon transfection trial on K562 & KAH126-MV2 <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

Overview

The neon transfection system is an electroporation instrument that allows you to transfect very small quantities of mammalian cells inside a conductive 10µL pipette tip. We were given a demo to try out in our lab. This is my attempt using K562 cells and the KAH126-MV2 plasmid.

Notes

I followed the procedure as outlined in the neon transfection kit. K562 parameters can be found here.

K562 culture density before resuspending in neon transfection buffer: 1.5E6 cells/mL. Cells were resuspended to 2E7 cells/mL in 60µL total.

KAH126-MV2 plasmid concentration is 809 ng/µL.

Volume of a.b.-free medium in each well of the 6-well plate: 2 mL.

For each well, I added 10µL of the resuspended cells to 1µL of plasmid or water in a sterile microcentrifuge tube.

Well layout:

  • Wells 1 through 4: treatment with plasmid
  • Wells 5 through 6: negative control (water)

The 6-well plate was then placed in the 37°C incubator. Will check fluorescence tomorrow for an estimate of efficiency.



Retrieved from "https://openwetware.org/mediawiki/index.php?title=Haynes_Lab:Notebook/RNA-seq_of_PcTF_Transfected_U2OS_%26_SK-N-SH_cell_lines/2015/04/23&oldid=882224"