Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/05/27: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
(Autocreate 2015/05/27 Entry for Haynes_Lab:Notebook/RNA-seq_of_PcTF_Transfected_U2OS_&_SK-N-SH_cell_lines)
 
No edit summary
Line 1: Line 1:
{|{{table}} width="850"
{|{{table}} width="850"
|-
|-
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Today's project is...</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Neon transfection of K562 cell line</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|-
|-
Line 8: Line 8:


==Overview==
==Overview==
Trying transfection of K562 again, this time using the Neon transfection system.
==Protocol==
# Set up a 12 well plate with 1 mL of antibiotic-free medium in each well. Place in the incubator.
# Spin down 5 mL of K562 culture and wash with PBS. Spin down again, resuspend in 250µL Resuspension Buffer R. Check density using hemocytometer and dilute to 1E7 cells/mL using Resuspension Buffer R.
# Follow the instructions on page 2 of the Neon Transfection System reference card to proceed.
Samples:
# pMax GFP, 1000 v, 50 ms, 1 pulse
# pMax GFP, 1350 v, 10 ms, 4 pulses
# pMax GFP, 1450 v, 10 ms, 3 pulses
# KAH126-MV2, 1000 v, 50 ms, 1 pulse
# KAH126-MV2, 1350 v, 10 ms, 4 pulses
# KAH126-MV2, 1450 v, 10 ms, 3 pulses
Treat samples in duplicate, for 12 samples in total.


<!-- Precede finished items with a checkmark &#x2713; -->
<!-- Precede finished items with a checkmark &#x2713; -->

Revision as of 12:22, 27 May 2015

Neon transfection of K562 cell line <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

Trying transfection of K562 again, this time using the Neon transfection system.

Protocol

  1. Set up a 12 well plate with 1 mL of antibiotic-free medium in each well. Place in the incubator.
  2. Spin down 5 mL of K562 culture and wash with PBS. Spin down again, resuspend in 250µL Resuspension Buffer R. Check density using hemocytometer and dilute to 1E7 cells/mL using Resuspension Buffer R.
  3. Follow the instructions on page 2 of the Neon Transfection System reference card to proceed.

Samples:

  1. pMax GFP, 1000 v, 50 ms, 1 pulse
  2. pMax GFP, 1350 v, 10 ms, 4 pulses
  3. pMax GFP, 1450 v, 10 ms, 3 pulses
  4. KAH126-MV2, 1000 v, 50 ms, 1 pulse
  5. KAH126-MV2, 1350 v, 10 ms, 4 pulses
  6. KAH126-MV2, 1450 v, 10 ms, 3 pulses

Treat samples in duplicate, for 12 samples in total.



Retrieved from "https://openwetware.org/mediawiki/index.php?title=Haynes_Lab:Notebook/RNA-seq_of_PcTF_Transfected_U2OS_%26_SK-N-SH_cell_lines/2015/05/27&oldid=886088"