Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/04/23: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Neon transfection trial on K562 & KAH126-MV2</span> | ||
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== | ==Overview== | ||
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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 [[https://www.lifetechnologies.com/content/dam/LifeTech/migration/en/filelibrary/cell-culture/neon-protocols.par.73680.file.dat/k-562-kidney.pdf 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. | |||
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Revision as of 18:44, 23 April 2015
 Neon transfection trial on K562 & KAH126-MV2
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OverviewThe 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. NotesI 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:
The 6-well plate was then placed in the 37°C incubator. Will check fluorescence tomorrow for an estimate of efficiency.
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