User:Karmella Haynes/Notebook/PcTF Genomics/2014/09/24: 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"| | |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]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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* Add 2 ml/well fresh media with 1% Pen/strep | * Add 2 ml/well fresh media with 1% Pen/strep | ||
* Grow cells at 37°C (overnight) | * 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 | |||
Latest revision as of 00:21, 27 September 2017
Pc-TF Genomics | Main project page Previous entry Next entry |
09/24/14
Transduction Day 2
Trouble-shooting. Maybe try this
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