<?xml version="1.0" encoding="UTF-8"?><ProtocolTitle xmlns="http://morstonmud.com/omero/schemas" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" description="Manufacturer's protocol for transfecting HeLa cells" elementName="Oligofectamine-Invitrogen" protocolFileName="Oligofectamine-Invitrogen.pro" protocolHasExpChildren="false" substepsCollapsed="false" version="3.0-3.0" xsi:schemaLocation="http://morstonmud.com/omero/schemas http://morstonmud.com/omero/schemas/PE.xsd"><FixedStep description="Oligofectamine has been used to transfect HeLa successfully (Elbashir etal 2001).&#10;The suggested (default) transfection conditions are provided as a starting point.&#10;&#10;" elementName="Introduction" substepsCollapsed="false"/><FixedStep description="Transfection Efficiency&#10;Transcription rate of gene of interest&#10;Protein Stability&#10;Efficicacy of the siRNA sequence used&#10;Cell line growth characteristics&#10;" elementName="Factors Affecting Knockdown Efficiency" substepsCollapsed="false"/><FixedStep elementName="General Guidelines" substepsCollapsed="true"><FixedStep elementName="Confluence 30-50%" substepsCollapsed="false"/><FixedStep elementName="Don't use antibiotics" substepsCollapsed="false"/><FixedStep description="Reduced serum medium (Cat no. 31985-062) to dilute Oligofectamine prior to complexing with siRNA." elementName="Use Opti-MEM I" substepsCollapsed="false"/></FixedStep><FixedStep elementName="Materials Needed" substepsCollapsed="true"><FixedStep description="or other mammalian cells. Must be healthy &gt;90% viable before transfection" elementName="HeLa cells" substepsCollapsed="false"/><FixedStep elementName="siRNAi (20 pmol / ul)" substepsCollapsed="false"/><FixedStep elementName="Oligofectamine" substepsCollapsed="false"/><FixedStep elementName="Opti-MEM I" substepsCollapsed="false"/><FixedStep elementName="24-well tissue culture plates" substepsCollapsed="false"/></FixedStep><FixedStep elementName="Conditions" substepsCollapsed="false"><NumberField default="50" description="30,000 cells per well is 50% confluent in 24-well plate" elementName="Cell density" requiredField="true" substepsCollapsed="false" units="%"/><NumberField default="3" elementName="Amount of Oligofectamine" requiredField="true" substepsCollapsed="false" units="ul"/><NumberField default="60" description="Stock should be 20 pmol/ul" elementName="Amount of siRNA" requiredField="true" substepsCollapsed="false" units="pmol"/><DropDownMenu default="48-hrs" dropdownOptions="24-hrs, 48-hrs, 72-hrs" elementName="Duration of transfection" requiredField="true" substepsCollapsed="false" value="24-hrs"/></FixedStep><FixedStep elementName="Proceedure" substepsCollapsed="true"><FixedStep description="One day before transfection, seed HeLa so they will be aprox 50% confluent at time of transfection" elementName="Plate HeLa" substepsCollapsed="false"/><FixedStep elementName="For each sample..." substepsCollapsed="false"><FixedStep description="mix gently" elementName="Dilute siRNA in 50 ul Opti-MEM" substepsCollapsed="false"/><FixedStep description="Incubate for 5 mins at room temp" elementName="Dilute 3ul Oligofectamine in 12 ul OptiMEM" substepsCollapsed="false"/><FixedStep description="Combine the two mixtures to give total volume of 68 ul. Incubate 20 mins at room temp" elementName="Mix siRNAi and Oligofectamine" substepsCollapsed="false"/></FixedStep><FixedStep description="Mix gently by rocking plate back and forwards" elementName="Add siRNAi:Oligofectamine to each well" substepsCollapsed="false"/><FixedStep description="in CO2 incubator for 24-72 hrs. Not usually necessary to remove complexes or change medium. However, can replace medium after 4-6 hours without loss of transfection activity." elementName="Incubate" substepsCollapsed="false"/></FixedStep></ProtocolTitle>