Editing Adenovirus AdEasy (section)

== Plaque Assay for Purification and Titration of rAdenoviruses  ==

'''Note:'''&nbsp; this only needs to be done for adenoviruses without GFP marker.&nbsp; In those which include GFP<font face="Arial Rounded MT Bold">marker, titration of infectious units (i.e., those resulting in expression of GFP) is simply determined by fluorescence microscopy of your favorite cell type.</font><br> 

1. Remove all but 2 ml per well of medium from&nbsp; 6-well plates containing 80% to 90% confluent 911 or 293 cells.&nbsp; Infect with appropriately diluted virus for 3 to 6 hours.&nbsp; Infect cells with 6 different dilution titers (e.g. 10<sup>-3</sup> to 10<sup>-8</sup>ml/well).<br> 

2. Prepare the overlay agar as following: autoclave 100 ml of 2.8% Bacto-Agar (Difco) and keep warm at 45<sup>o</sup>C water bath. For 100 ml overlay: 

{| cellspacing="2" cellpadding="2" border="1"
|-
| <br> 
| Stock Conc 
| Final Conc. 
| ml/100 ml overlay
|-
| 2 X BME (GIBCO) 
| 2 X 
| 1X 
| 50.0
|-
| HEPES 
| 1.0M <br> 
| 20.0mM 
| 2.0
|-
| MgCl2 
| 1.0M <br> 
| 12.5mM <br> 
| 1.25
|-
| FBS 
| 100.0% <br> 
| 10.0% 
| 10.0
|-
| Pen/Strep 
| 100 X 
| 1 
X&nbsp; <br> 

| 1.0
|-
| colspan="4" | mix well and incubate at 37<sup>o</sup>C bath.
|-
| Bacto-Agar 
| 2.8% <br> 
| 1.0% <br> 
| 36.0
|}

Mix well and swirl at 37<sup>o</sup>C water bath, add 4 ml/well for 6-well plate. 

'''Note''':&nbsp; If you want to melt 2.8% agar stock,&nbsp; microwave or heat it in boiling water bath.&nbsp; Prepare 50 ml overlay at a time. To prevent the pre-drying of agar before plaque formation, it is important to add sterile PBS or Hankâs medium in the space between wells, and wrap the plates with SaraWrap.<br> 

3. Return the plates to 37<sup>o</sup>C CO<sub>2 </sub>incubator.&nbsp; On days 5 - 7, overlay 2 to 3 ml agar containing nuetral red (from 100x stock, available from GIBCO-BRL) to each well.&nbsp; Plaques should be visible 16-30hrs after the neutral red overlay. (In general, plaques should form in 911 cells in 4 or 5 days and in 293 cells after 5-7 days).<br> <br> 

4. Pick 5 to 10 well-isolated plaques for each recombinant by punching out agar plugs with a sterile pasteur pipet. Store agar plugs in 200µl HBSS medium. Perform freeze/thaw/vortex cycle 4 times. Spin samples briefly.<br> 

5. Infect one well of 6-well plate of 911 or 293 cells (about 80-90% confluency at time of infection) with 50-70% virus sup of each plaque for 3 to 5 days (depending on the titer of individual plaque).&nbsp; <br> 

Presence of recombinant virus can be confirmed by Western blot or PCR.<br> 

6. Collect viral supernatant using four cycles of freezing/thawing, as described earlier in this guide. The virus sup can be used to further infect T-25, and then T-75 flasks, for large scale preparation.<br> <br> 

[[Category:Protocols]]