Pelling:Protocols/Stable Line

From OpenWetWare
Jump to: navigation, search

<owwmenu font="arial, helvetica, sans-serif" bold="1"

       color="black" bgcolor="white" hovercolor="black"
       bghovercolor="gray" topfontsize="10" fontSize="8" pagewidth="930"
   Lab Supplies=Pelling:Lab Supplies
   Lab Safety and MSDS's=Pelling:Lab Safety


*** UNDER CONSTRUCTION - in the meantime see Pamela for a protocol ***

Making Drug Stocks

  • If you already know what concentration you want to use for selection, skip to “Making Drug+DMEM Stock”.
  • Measure out and dissolve powdered drug into complete DMEM. Suggested stock concentration varies depending on the drug used. Much less than 1ml is enough for a kill curve, and later you need to make drug+DMEM stock.
Drug [Stock]
G418 200 mg/mL
Puromycin 2.5 mg/mL
Hygromycin 200 mg/mL
  • Cover the tube in aluminum foil to prevent light exposure (optional).

Making a Kill Curve

  • Kill curves are suggested for a new shipment of drug or for cell lines that do not have a lot of literature on them. Sometimes you can skip this step if you can find lots of literature values. For example, some protocols say G418 will work on HeLa cells at 400-500μg/mL.
  • Plate freshly split cells onto a multi-well plate. You need approximately 12 wells for each drug. The following table gives suggested amounts and final concentrations for a 12-well plate filled with 1ml in each well.