Q: Why did I get no colonies when I cotransform the PmeI-cut shuttle plasmids with pAdEasy backbone vectors into BJ5183 cells?

A: In most cases, this is a result of bad competent cells. Make sure your BJ5183 cells are highly competent. Also, you should always include a positive control plasmid in your transformation. Another common cause of this problem is the quality of your miniprep DNAs. As mentioned in the protocol, the pAdEasy vectors should be CsCl-banded.  It is very difficult to get good and consistent quality of the shuttle vector DNA from most commercial miniprep kits. A lot nicked DNA will be generated from these prep kits. In our experience, we get the most reliable and consistent results when the conventional alkaline lysis miniprep procedure (see Protocol section) is used. Majority of the miniprep DNA from this procedure are in supercoiled form, which is important for correct homologous recombination.
 

Q: What are the best ways to prepare good competent BJ5183 cells?

A: The most critical step for using the AdEasy system is to make good competent BJ5183 cells. These cells tend to be less efficient than what for transformation. To make good competent BJ5183, you should: 1). eliminate any contaminating strains by growing the cells in the presence of streptomycin; 2). chill the cells longer on ice (2-4hours); 3). thoroughly wash the cells with 10% glycerol (2-3 times); and most importantly, keep the cells concentrated (e.g. one liter culture yields <2 ml competent cells).
 

Q: How can I best select potential recombinants?

A: Always pick up the smallest colonies. However, this can be very difficult if: a) you have too many colonies on the plates; b). or bacterial cells are not spread evenly over the plates.
 

Q: Why do I have too many colonies and have a difficult time picking up true recombinants?

A: It is likely that your PmeI digestion did not work well. Check portion of your digestion on agarose gel. Gel purifying the linearized DNA is always an option, although we have found that gel purified fragments sometimes decrease the transformation efficiency.

The background can also be significantly reduced by eliminating the incubation of the transfected cells at 37 ^oC. Practically, folks in our lab have minimized this problem by directly plating the bacterial cells after electroporation. For unclear reasons, this seems to reduce the background colonies without much affecting the recombinants.

In most cases though, if you are getting too many colonies (e.g. >100 clones per transformation), it usually indicates that too much shuttle vector DNA is used for the co-transformation. You should titrate down the shuttle vector DNA to the range of 100-500ng. It is also very helpful to miniprep the shuttle vector DNA using the alkaline lysis method (instead of commercial miniprep kits).
 

Q: I was initially able to get recombinants; and all of sudden, I am no longer able to get any recombinants?

A: It could be due to different batches of competent BJ5183 cells. It could also be a result of contamination of BJ5183 cells with other strains. To eliminate this possibility, you should grow the BJ5183 cells in the medium containing 30ug/ml streptomycin.
 

Q: Can I use the chemical transformation procedure instead of electroporation?

A: It is not recommended because the efficiency is expected to be much lower.
 

Q: Can I PCR screen for the recombinants using cross-junction primers?

A: It is possible. But you have to maintain low PCR cycle numbers, e.g. 20-25 cycles. We tried initially and got a lot false positives.
 

Q: Can I establish a BJ5183 strain containing the pAdEasy-1 plasmid to avoid co-transformation?

A: We have initially thought about using this strategy, but concerned about the potential rearrangement of the backbone vector in the bacterial cells.
 

Q: Why is my digestion pattern of the vectors different from that of the Fig. 3 of the PNAS paper?

A: You should compare your digestion results with the maps and sequences posted in this website. Some of the results in the PNAS paper refered to the earlier versions of the vectors.
 

Q:   What cells should I use for viral production and amplification?

A:   293 cells are satisfactory for most applications.   911 cells are more efficient for viral production and form plaques sooner, but grow a bit slower than 293 cells.  Thus, we routinely either use 293 cells for the entire process, and only use 911 cells for special case.  See Cells for producing adenoviruses  for information about obtaining 293 and 911 cells.
     For preparation of viruses with inserts larger than 7.5 kb in size, an E4-producing line is required.  See Cells for producing adenoviruses  for information about E4-producing cell lines.
 

Q: I do not see apparent plaques or CPE after the initial transfection. Did I do anything wrong?

A: In most cases, you probably will not see any apparent plaques formed or drastic CPE even at 10 days after the transfection. We have never failed to generate viruses even though we do not observe plaques or CPE. Whether you see plaques or not probably depends on the transfection efficiency, which only affects the initial viral titer. On the other hand, if you use AdTrack derivatives, you should observe some comet-like clusters under fluorescence microscope after 5 to 7 days of transfection.
 

Q: Should I ever plaque-purify my viruses?

A: AdEasy system usually generates very homogeneous recombinant viruses. We have found that at least  95% of  the cells infected with a GFP/lacZ virus made with this system expressed both transgenes. Thus, the viruses should be pure enough for most experiments. For in vivo use, plaque purification may be desirable
 

Q: What should I do differently if  I use the AdEasy-2 system?

A:  Use these only if you have a very large insert (7.5 - 10 kb).  Almost the same as the AdEasy-1 system except: 1). use pAdEasy-2  for cotransformation of BJ5183 cells; 2). Use an E4-producing line.  If you use 911E4 cells (see Cells for producing adenoviruses) for this purpose, maintain them  in complete DMEM containing 0.4mg/ml geneticin, 0.1mg/ml hygromycin B, and 50 ng/ml doxycyclin. When you transfect the cells, doxycyclin should be included in the OptiMem medium, and  removed from the medium 24 hours after transfection.
    We and others have found that the AdEasy-2 system is generally more challenging mostly because the E4 packaging cells gradually lose their expression of the E4 gene over passages.
 

Q: Can I use the same shuttle plasmids for both AdEasy-1 and AdEasy-2 systems?

A:  Yes. There is a second homologous recombination site between the two pBR322 Ori sequences. In this case, PacI digestion of the recombinants should yield a 4.5 kb fragment (instead of 3.0 kb). They are equally efficient for viral production.
 

Q: Is it possible to remove the CMV promoter or GFP cassette from the shuttle vectors?

A: It is very difficult because these cassettes were cloned by blunt-ending ligations to ensure more cloning sites in the polylinker. However, you should be able to construct your desired vector by using the pShuttle vector.
 

Q: Is the transgene expression level affected by using two identical CMV promoters?

A: The expression of both genes does decrease to certain degree (e.g. 3- to 5-fold lower compared with single cassette vector). But CMV is such a strong promoter and the expression levels are high enough for most experiments. 
 

Q: Why do I only get a very faint adenoviral band in the CsCl gradient?

A:  This usually results from less than ideal amplification in the last round.  If you infect 293 cells at too low titer (e.g. no obvious cell lysis after 4-5 days) or too high titer (e.g. cells lysed too soon, like less than 2 days), both will produce a faint band or no band on CsCl gradient preparations.
 

Q: Can I dialyze the CsCl and keep the viruses at -20^oC?

A: You should not store adenovirus this way because the viruses are more stable in high salt conditions. Always dialyze the viuses right before you use them.
 

Q: Is there a simple and quick way to dialyze the CsCl-banded viruses?

A: Many people use commercially available microdialysis devices or spin columns. We have used a simple method called agarose-tube dialysis (see Protocol section).
 

Q: Are the full-length sequences for the vectors available?

A: Yes. They are included in this website (see Vector Maps and Sequences section). Although there are some small sequence gaps at the cloning junctions, they should provide valuable information for primer designs and restriction digestions.

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