I've been having difficulties inducing a proper amount of lung nodules in a KRAS-driven (KrasLSL-G12D) conditional mouse lung cancer model following this protocol:

Conditional mouse lung cancer models using adenoviral or lentiviral delivery of Cre recombinase

https://www.nature.com/articles/nprot.2009.95

This is the Nature Protocol paper from Tyler Jacks lab that I have been using as a reference

Reagents

• MEM (Sigma catalog #M-0268)
• 2 M CaCl2
• Adenovirus - University of Iowa (VVC-U of Iowa-5 Ad5CMVCre)*

Add 2.5 uL of Ad5CMVCre to 121.9 uL of MEM and mix well.

Add 0.6 uL of CaCl2 and mix well.

Let this mixture sit for ~20 minutes before use.

I have been using 2.5 x10E7 pfu for my experiments. Here are my questions:

1. When making the virus prep, is it a homogeneous solution after calcium phosphate precipitate formation? I wonder if one needs to flick the tube or pipette to mix it well after sitting on ice for 20 minutes and before giving it to the mice.

2. Can I make a "master mix" virus prep for all mice dosed on the same day? Or should I prepare one tube per mouse?

3. Is there a specific reason one must use 2M CaCl2 when making virus prep? Because sometimes 0.6 uL could be hard to pipette.