User:Karmella Haynes/Notebook/PcTF Genomics/2014/09/24

Pc-TF Genomics

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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