Generating and Amplifying Adenoviral Constructs: Difference between revisions

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 #Add an equal volume of 2X Virus storage buffer and store at -80
 #Titer the virus using a kit.
+===desalting of CsCl purified adenovirus using PD-10 desalting columns (17-0851-01 GE Healthcare)===
+# Cut column, discard storage solution and wash X5 with ~5ml PBS
+# Add virus sample (up to 2.5 ml)
+# Elute with 5 ml PBS, discard first 20 drops and collect 10 drop fractions into 10 tubes.
+# OD 260 fractions in nanodrop, virus is in middle fractions (5-7 in my extraction). Good virus yield is >3 OD.
+# Add 10% glycerol and store at -80.
+===Tail Vein Injection===
+# Insert mouse to restriction device. Carefully warm tail with lamp/warm water, inject 0.1 OD/mouse (0.15 for HFD mouse) using insulin syringe.
+# Injected mice are housed in biohazard mouse room for 3 days (need bright-pink barcode stickers and water bottles).
+# Adenovirus is detected in liver for at least 2-3 weeks, shRNA has maximal effect at 4-6 days, usually gone in 10 days.