User:Karmella Haynes/Notebook/PcTF Genomics/2014/09/24: Difference between revisions

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==mm/dd/yy==
==09/24/14==
<!-- Precede finished items with a checkmark &#x2713; -->
<!-- Precede finished items with a checkmark &#x2713; -->
* Line item 1
* Transduction Day 2: change media
 


----
----
'''Line item 1'''<br>
'''Transduction Day 2'''<br>
> Samples
* Brought 25 mL aliquots of media for each cell line to the LaBaer lab.
* Checked GFP control wells (LaBaer scope has no RFP filters)
** Plate 1: U2OS, ~90% GFP expression, at least ~two doubling
** Plate 2: SK-N-SH, ~90% expression, slow cell growth
** Plate 4: K562, <font color="red">no GFP expression. Did not continue next steps for this plate</font>
* Remove media
* Add 2 ml/well fresh media with 1% Pen/strep
* Grow cells at 37°C (overnight)


{| class="wikitable" border="0" cellspacing="3" <!-- Rxn. table -->
|-valign="top"
| <u>Reagent</u> || <u>Volume</u>
|-
| reagent 1 || # μL
|-
| reagent 2 || #
|-
| reagent 3 || #
|-
| reagent 4 || #
|-
| dH<sub>2</sub>O || #
|-
| &nbsp; || # μL
|}


--> Reaction conditions
Technical hint from Research Gate
* In our lab, we concentrate our lentivirus after production using PEG-precipitation. This increases the concentration 50-100x. '''Then, we "spinoculate" K562 cells with a small amount of virus in an eppendorf tube. Typically, this means putting 50,000 - 100,000 K562 cells in an eppendorf tube, adding 1-2 uL of lentivirus, adding polybrene to 4 ug/ml, and centrifuging for 30 min at 800 x g (at 32 C or room temperature).''' If your lentivirus is of good quality, this method gives very high infection rate (normally I get greater than 90%...close to 100).
** Mark Duncan, Northwestern U.


Trouble-shooting. Maybe try this
* Put 80k/1 mL K562 cells + Polybrene + 0.4 mL viral aliquot into 15 mL conicals
* Spin, according to Ganzalez protocol (9/23/14)
* Resuspend and plate total volume in 6-well plate




Revision as of 07:16, 28 September 2014

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09/24/14

  • Transduction Day 2: change media


Transduction Day 2

  • Brought 25 mL aliquots of media for each cell line to the LaBaer lab.
  • Checked GFP control wells (LaBaer scope has no RFP filters)
    • Plate 1: U2OS, ~90% GFP expression, at least ~two doubling
    • Plate 2: SK-N-SH, ~90% expression, slow cell growth
    • Plate 4: K562, no GFP expression. Did not continue next steps for this plate
  • Remove media
  • Add 2 ml/well fresh media with 1% Pen/strep
  • Grow cells at 37°C (overnight)


Technical hint from Research Gate

  • In our lab, we concentrate our lentivirus after production using PEG-precipitation. This increases the concentration 50-100x. Then, we "spinoculate" K562 cells with a small amount of virus in an eppendorf tube. Typically, this means putting 50,000 - 100,000 K562 cells in an eppendorf tube, adding 1-2 uL of lentivirus, adding polybrene to 4 ug/ml, and centrifuging for 30 min at 800 x g (at 32 C or room temperature). If your lentivirus is of good quality, this method gives very high infection rate (normally I get greater than 90%...close to 100).
    • Mark Duncan, Northwestern U.

Trouble-shooting. Maybe try this

  • Put 80k/1 mL K562 cells + Polybrene + 0.4 mL viral aliquot into 15 mL conicals
  • Spin, according to Ganzalez protocol (9/23/14)
  • Resuspend and plate total volume in 6-well plate



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