Cloning genes into the AdZ vectors and making virus Click here to download the protocol (pdf format) for inserting genes with any tag into the AdZ vectors. Note that the vector numbers are different to those in the original publication - the original work used vectors in which the genome was derived from the AdEasy vectors. The new vectors contain a newly derived & sequenced Ad5 genome, but the expression cassettes etc remain identical to those in the original paper.
Using AdZ-CRISPR vectors. Click here to download a protocol for using the AdZ vectors to knock genes out with CRISPR.
Growing RAds If you're familiar with growing RAds then carry on as usual. This is the protocol we use for reconstituting RAds from the AdZ BACs, and growing them up.
Titering viruses When it comes to titering the virus you've made, you can use any method (counting plaques, limiting dilution, measuring OD). However with the exception of measuring OD, these require the cells to stay alive in a well for a good few weeks & we've found that the 293TREx cells don't last well enough. We therefore use a simple immunofluorescence protocol to get titres. Click here to download the protocol. Alternatively, if you don't have a toxic transgene, you can use any of the usual titering methods in 293/911/PERC6 cells.
General Recombineering Quite a few people have asked for protocols for inserting the sacB cassette elsewhere in the genome, in order to make their own modification. This protocol is the protocol I use for inserting & then removing the cassette in order to modify the backbone.