Can the bacluovirus infect mammalian cells? How about Drosophila cells?

Product FAQ

Answer

Yes, baculovirus can infect mammalian cells, although only at very high titers. Baculovirus works best in liver cells. However, there is no danger of cross-contamination unless the cells are directly infected with the high-titer stocks. Bacuolvirus can infect Drosophila cells; however, it will not replicate in these cells. The promoters used to drive expression of your gene in a typical baculovirus system are both late promoters and require earlier proteins from the baculovirus genome. Thus, they will not work in S2 cells since the early proteins are not made.

Answer Id: E9433

Was this answer helpful?

Yes
No
Thank you for your response

Can a P3 viral stock be used to generate more viruses? How about a P4 or P5 stock?

Product FAQ

Answer

Yes, the same protocol used to make your P2 viral stock can be used to make a P3, P4, or P5 viral stock. We don’t recommend making the stock higher than P5, as more defective interfering particles will be produced and a decrease in protein expression level will occur.

Answer Id: E9434

Was this answer helpful?

Yes
No
Thank you for your response

Can an old low-titer stock be used to make a high-titer stock?

Product FAQ

Answer

If this lower-titer stock is a P1 or P2 stock, a viral amplification protocol can be used. If the low-titer stock was once a high-titer stock, but has dropped titer due to age or the stock was propagated many generations, then it may be necessary to regenerate the high-titer stock. If the high titer stock is >P5, then there may be an excessive amount of defective interfering particles that infect cells but do not properly replicate or produce protein. If the existing stock is plated out and a fresh plaque is re-isolated (DIPs do not form plaques), a new high-titer stock can be established.

Answer Id: E9435

Was this answer helpful?

Yes
No
Thank you for your response

I’m yielding no plaques from my baculovirus plaque assay. What are the possible causes for this?

Product FAQ

Answer

The kinetics of infection may be slower than expected. Observe plates until the 8-9th day after infection. If no plaques appear, investigate the following:

- If the cells are not healthy, then poor-quality or no plaques can result. Ideally, cells should be in mid-log phase and have a viability of greater than 90%. Cells should double at least once before infection stops growth. Ensure that the correct amount of cells was used at ~70% confluency.
- The viral replication cycle can be inhibited due to poor nutritional and physical conditions of the cell.
- The temperature of the agarose is also crucial. After overlaying the agarose, the plates should be left untouched for 1 hour for the agarose to completely solidify.
- Excessive condensation during incubation at 27 degrees C can inhibit plaque formation-remove paper towels or open the container containing plates as soon as condensation appears.
- The viral titer is too low: Use a higher viral titer. You may need to re-infect your cells and collect a higher titer of your viral stock.

Answer Id: E9465

Was this answer helpful?

Yes
No
Thank you for your response

I’ve infected my cells and see large polyhedra in one cell and smaller polyhedra (more numerous) in a neighboring cell. Is this normal?

Product FAQ

Answer

Yes, cells are infected with wild-type virus individually and will develop polyhedra at different rates until all the cells in the flask are infected. The polyhedra in cells will form in approximately 3-4 days, differing in size and number until they reach their maximum capacity and burst the cell, releasing tiny particles of virus into the medium.

Answer Id: E9473

Was this answer helpful?

Yes
No
Thank you for your response

I think I’ve added too much virus. How important is the MOI? What happens if too much virus is used?

Product FAQ

Answer

An MOI of 5-10 is typically used. If too much virus is added, unfortunately the cells die too soon and the protein expression level goes down.

Answer Id: E9466

Was this answer helpful?

Yes
No
Thank you for your response

Are the Gateway™ attB1 and attB2 sites the same as the attB site used for recombination into E. coli by bacteriophage lambda?

Product FAQ

Answer

The Gateway™ attB sites are derived from the bacteriophage lambda site-specific recombination, but are modified to remove stop codons and reduce secondary structure. The core regions have also been modified for specificity (i.e., attB1 will recombine with attP1 but not with attP2).

Answer Id: E3201

Was this answer helpful?

Yes
No
Thank you for your response

I’m getting mostly white/wild-type plaques instead of blue/recombinant plaques. What am I doing wrong?

Product FAQ

Answer

This is typically an indication of poor homologous recombination. Check the plasmid/linear DNA ratio you used. If there are some blue plaques, however, expand those viruses and check for their protein. In our experience, they are correct, even if they were in relatively low abundance.

Answer Id: E9474

Was this answer helpful?

Yes
No
Thank you for your response

Can PCR primers be tailed directly with attL sites for direct recombination into the destination vector?

Product FAQ

Answer

No, this is not really feasible due to the fact that the attL sequence is approximately 100 bp, which is too long for efficient oligo synthesis. Our own maximum sequence length for ordering custom primers is 100 nucleotides. In contrast, the attB sequences are only 25 bp long, which is a very reasonable length for adding onto the 5' end of gene-specific PCR primers.

Answer Id: E3212

Was this answer helpful?

Yes
No
Thank you for your response

Is there a way to make baculovirus plaques more visible or distinctive?

Product FAQ

Answer

You can stain the monolayer with neutral red or MTT to make the plaques more visible. Alternatively, you can allow the plates to develop for a few days longer (2-5 days on average) at room temperature to increase the contrast in recombinant plaques. However, the plaques stained with neutral red cannot be used for plaque purification and viral amplification.

Answer Id: E9427

Was this answer helpful?

Yes
No
Thank you for your response

I see what looks like small plaques, but they are too difficult to visualize. What could be the problem?

Product FAQ

Answer

Too many cells were seeded; we recommend seeding 8 x 10e5 cells per well for a 6-well plate.

Answer Id: E9467

Was this answer helpful?

Yes
No
Thank you for your response

Where is the ATG relative to the 5' attB site in a Gateway™ expression clone?

Product FAQ

Answer

This depends on whether you are expressing a fusion or a native protein in the Gateway™ destination vector. For an N-terminal fusion protein the ATG will be given by the destination vector and it will be upstream of the attB1 site. For a C-terminal fusion protein or a native protein, the ATG should be provided by your gene of interest, and it will be downstream of the attB1 site.

Answer Id: E3202

Was this answer helpful?

Yes
No
Thank you for your response

What has happened when I see blue colonies? How about colonies which are blue in the center and white on the edges?

Product FAQ

Answer

In the case of a blue colony, the E. coli has the bacmid and the plasmid in it, allowing the cells to survive the selection process. However, because the transposition has not occurred, the LacZ gene is not disrupted. For bulls-eye colonies, this indicates that the transposition took place when the colony was growing. Re-streaking for an isolated clone from the white portion of the mixed colony should yield some colonies where transposition occurred.

Answer Id: E9475

Was this answer helpful?

Yes
No
Thank you for your response

What size targets can be amplified by the SuperScript™ One-Step RT-PCR Systems?

Product FAQ

Answer

The SuperScript™ III One-Step RT-PCR System with Platinum™ Taq DNA Polymerase is recommended for amplifying RNA targets up to 4.5 kb and the SuperScript™ II One-Step RT-PCR System with Platinum™ Taq DNA Polymerase is recommended for amplifying RNA targets up to 3.5 kb. Even though the SuperScript™ II and III One-Step RT-PCR Systems with Platinum™ Taq High Fidelity can amplify smaller targets, it is recommended for amplifying RNA targets from 1 kb up to 9 kb.

Answer Id: E3213

Was this answer helpful?

Yes
No
Thank you for your response

Can I sequence a peptide that is acetylated or biotinylated?

Product FAQ

Answer

No. These groups effectively block the N terminus.

Answer Id: E1267

Was this answer helpful?

Yes
No
Thank you for your response