MultiColi/MultiBac User Tips

• Unlike most pET vectors, MultiColi and Multibac vectors do not contain LacI. We have observed that this can be problematic when expressing toxic genes.
• Sequence and Ligation Independent Cloning (SLIC) as described in the user manuals is recommended.
• Donor vectors contain a R6Kγ conditional origin of replication, which require propagation in pir⁺ host strains. CSB has both high copy (pirHC) and low copy (pirLC) strains.
• Although I have used the spectinomycin resistant plasmids pDS (MultiColi) and pIDS (MultiBac) successfully, I had better luck cloning into the other donor vectors. In pirHC, pDS and pIDS tended to produce lawns, whereas in pirLC, they produced no transformants.
• The efficiency of Cre fusion drops when more than 2 vectors are fused, so running reactions sequentially is recommended.
• Fusing equimolar amounts of each vector decreases the chance of integrating more than one copy.
• Restriction digestion is the best way to characterize Cre-fused constructs.
• Fusing more than 3 vectors is not recommended because the product is almost impossible to characterize. (Three vectors give 2 possible fusion products; four vectors give 6.)
• We did not have any luck using bacmid isolated from DH10 Multibac cells so the CSB no longer stocks them.
• DH10 EmbacY cells (Kan/Chlor/Tet) have the viral LoxP site occupied with YFP under the control of polyhedrin promoter. See Trowitzsch, et al. J Struct Biol. 2010 Oct;172(1):45-54. We successfully used YFP fluorescence to monitor virus performance.