User Manual for Ready-to-use AAV Products
Contents
1. VECTOR BIOLABS The Gene Delivery Company User Manual for Ready to use AAV Products CONTENTS AND STORAGE AAV stocks are supplied in liquid form at indicated titer The storage solution PBS 5 glycerol Store at 80 C If desired aliquot viral stock upon arrival and store those aliquots at 80 C freezer immediately DO NOT FREEZE AND THAW REPEATEDLY IMPORTANT GUIDELINES Follow these guidelines when performing infections 1 Prepare virus containing media Thaw viral stock at either room temperature or on ice Add desired amount of virus to growth media to achieve the desired MOI AAV GC particles to be used MOI multiplicity of infection of cells to be infected e g If you intend to infect 1 million cells using MOI of 1 000 you need 1 000 x 1 000 000 10 GC for the infection If the original stock is 10 GC ml then you will need 0 1 ul of the original stock for the dilution 2 Infecting cells with virus Remove the original cell culture media and add the above AAV containing media to cell culture Below is a general guideline for the amount of media used 24 well plate 0 2 0 3 ml 12 well plate 0 5 0 8 ml 6 well plate 1 1 5 ml well 60mm plate 3 4 ml plate 10cm plate 8 12 ml plate Incubate cells with the virus containing media for 6 12 hours or as long as you wish Optional you could remove virus containing media and replace it with fresh desired media The appropriate amount of viruses used2. for infecting cells is critical for the outcome of your experiments The goal is to get 100 of infection without causing any undesired effects The optimal concentration differs dramatically between cell types for different serotype of AAV For AAV serotype 2 a range of 2 000 10 000 MOI is used for many cell lines HEK293 CHO Hela HepatA etc but up to 1 000 000 MOI may be used for many other cells To determine this optimal concentration of virus for your study you could conduct pilot testing in your cell line by using reporter AAV like AAV GFP Note 1 AAV stock can be added directly to cells in culture medium in the presence or absence of serum 2 It is not necessary to remove viruses change or add medium following infection although viruses can be removed after 6 12 hours post infections 3 It can take 3 7 days after the AAV infection to detect the gene over expression Date of revision April 20 2011 3624 Market Street Suite 5E Philadelphia PA 19104 Tel 877 BIOLABS Fax 215 525 1112 info vectorbiolabs com
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