User:Cassandra M Barrett/Notebook/Haynes Lab/2015/08/28

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Analyzing Cells with Flow Cytometry and Preparing Genomic DNA for Rene

Purpose: to analyze fluorescence of cells for Rene's time point experiment and set up their DNA for extraction

Methods:

Two cell types: one treated with puramycin and the other treated with doxacyclin Media: DMEM complete

Aspirate media out of wells with p200 tip on aspirator. Gently drop in 0.5mL PBS to wash Aspirate out PBS Add 0.2mL trypsin to each well (2-3 drops, just enough to cover) to loosen cells Label 6 tubes (dox 72hr 1-3 and puro 72h 1-3) Create a 2.6mL aliquot of media in a conical tube Aliquot 400uL of the media into each well Remove cells and media into appropriate tube

Spin tubes 3mn at 0.2 Get ice Put PBS no TC on ice Remove supernatant to top of pellet (abut 50uL left) Was with 500uL PBS Spin again at 0.2 for 3min

Get 6 strainers and label (d1-d3, p1-p3) Heat block to 56

Remove PBS until top of pellet Resuspend in 400uL PBS Filter 200uL Perform Flow Cytometry on filtered sample (10,000 events in gate)

Flow notes: events/sec in water should be less than 10, dilute sample if events/sec are over 500 Had trouble with low events/sec because of old culture... Had to add more PBS a couple times just to have enough for cells to flow


Genomic DNA Prep

Qia DNA mini kit

Add 20uL proteanase K, gently spin Add 200uL of buffer AL Vortex 15s Heat at 56C for 10min Place in Rene's box in -20C freezer

Be careful of reagents!