Editing Adenovirus Production (section)

== Vector Linearization with PacI ==

1.Digest 5 ul of GOI in pAd5-CMV-MCS shuttle vector and pAd5B (Clone #415, backbone) vector (4.5 ug) with '''PacI.'''

'''Reaction A'''

GOI in pAd5-CMV-MCS miniprep DNA: 5 ul

pAd5B vector (#415) 1 ug (~2 ul of maxiprep DNA)

PacI, 1 ul

Buffer 1, 2&nbsp; ul

10X BSA, 2 ul

Water to 20 ul



Incubate at 37 oC for 2 hour or room temperature O/N.

'''After digestion'''

2. Optional: Run 2&nbsp;ul of each digested DNA and undigested DNA on a 0.8% agarose gel to confirm the completion of PacI digestion





*<strike>Precipitate pAd5B vector with 3M KAc + isopropanol, wash with 75% EtOH, dry in biosafety hood, dissolve in H<sub>2</sub>O.</strike>
*<strike>or:</strike> Purify DNA with Omega miniprep spin column, Elute DNA with 50 ul sterile dH<sub>2</sub>O. Store the digested DNA at -20 °C.