User:Karmella Haynes/Notebook/PcTF Genomics/2014/09/23: Difference between revisions

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Pc-TF Genomics</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Pc-TF Genomics</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"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
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'''Transductions'''<br>
'''Transductions'''<br>
Note: cells are in standard complete medium
# Plate 1: U2OS
# Plate 1: U2OS
# Plate 2: SK-N-SH
# Plate 2: SK-N-SH
# Plate 4: K562 b (1 mL) - no one was around to consult on whether I should pellet the 2mL and replate
# Plate 4: K562 b (1 mL) - no one was around to consult on whether I should pellet the 2mL and replate


Abbreviated protocol (from Seron Eaton)
Wells (for all plates) - added 400 μL virus
* Add  
# well 1 - GFP
# well 2 - KAH160/015 (2010)
# well 3 - KAH160/015 (2014)
# well 4 - KAH165/015 (2010)
# well 6 - KAH165/015 (2014)
# well 7 - KAH170/015 (2014)
 
 
Day 1: Abbreviated protocol (from Laura Gonzalez)
* Retrieve frozen aliquots of harvested virus from -80C freezer (just outside TC room)
* Thaw and quick-spin to get virus off of the inside of lid
* Biohazard set-up: 10 mL Wescodyne waste conical (50 mL), 10 mL Wescodyne rinse
* Remove 1mL of media from each well (except for K562)
* Add 5ul of 2.2mg/ml Polybrene (thawed) to each well. Note: final concentration of Polybrene in well will be 8μg/ml after 400 μL viral aliquot is added
* Spin at 2250 rpm for 25 minutes
* Place the plates in the incubator (37°C) overnight.




Latest revision as of 00:21, 27 September 2017

Pc-TF Genomics Main project page
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09/23/14

  • Transductions: U2OS, SK-N-SH, K562

Transductions
Note: cells are in standard complete medium

  1. Plate 1: U2OS
  2. Plate 2: SK-N-SH
  3. Plate 4: K562 b (1 mL) - no one was around to consult on whether I should pellet the 2mL and replate

Wells (for all plates) - added 400 μL virus

  1. well 1 - GFP
  2. well 2 - KAH160/015 (2010)
  3. well 3 - KAH160/015 (2014)
  4. well 4 - KAH165/015 (2010)
  5. well 6 - KAH165/015 (2014)
  6. well 7 - KAH170/015 (2014)


Day 1: Abbreviated protocol (from Laura Gonzalez)

  • Retrieve frozen aliquots of harvested virus from -80C freezer (just outside TC room)
  • Thaw and quick-spin to get virus off of the inside of lid
  • Biohazard set-up: 10 mL Wescodyne waste conical (50 mL), 10 mL Wescodyne rinse
  • Remove 1mL of media from each well (except for K562)
  • Add 5ul of 2.2mg/ml Polybrene (thawed) to each well. Note: final concentration of Polybrene in well will be 8μg/ml after 400 μL viral aliquot is added
  • Spin at 2250 rpm for 25 minutes
  • Place the plates in the incubator (37°C) overnight.



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