After addition of the agarose overlay to my cells, they look like crescents or are granular. What happened?

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Answer

The agarose overlay was too hot. After addition of the agarose overlay, cells should still be round and healthy.

Answer Id: E9469

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What might cause plates to turn blue along with blue plaques?

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Answer

There are a few things that can turn plates blue:

- Too much virus when plating. Try a higher dilution.
- Cells are being singed when plated with hot melted agarose. This lyses the cells and releases lacZ into the agarose, turning it blue. Double-check plating temperatures. If plates are too wet, the blue can diffuse.

Answer Id: E9470

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Could you please provide me with a protocol overview for the BaculoDirect™ system?

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Answer

Begin your BaculoDirect™ experiments by cloning in your gene of interest into your Gateway™ Entry Vector, followed by the LR Clonase™ reaction into the BaculoDirect™ vector. Transfect this vector into your cells and grow for 3 days. On the 4th day, collect P1 viral stock. Re-infect cells, and grow for 3 days. On the 7th day, collect P2 viral stock. Infect cells, followed by harvesting of protein and purification on the 10th day.

Answer Id: E9415

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I forgot to add Bluo-gal to my plates. Can I add it later?

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Answer

On the day you intend to pick plaques, make a solution of Bluo-gal in DMSO at 20 mg/mL. Add 50 μL per plate and spread with a glass spreader under sterile conditions. Wait 30-60 min, and your plaques should turn blue.

Answer Id: E9471

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What cell lines do you recommend using with the BaculoDirect™ system?

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Answer

We recommend using Sf9 or Sf21 cells to generate high-titer viral stocks. We do not recommend using High Five™ cells to generate viral stocks due to lower transfection efficiency. Once you have generated your high-titer viral stocks, you can use Sf9, Sf21, High Five™, or Mimic™ Sf9 cells for protein expression.

Answer Id: E9416

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

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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

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What does viral infection look like in early, late, and very late stages?

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Answer

Please see the description below of the different stages of viral infection:

Early
- Increased cell diameter-a 25-50% increase in the diameter of the cells may be observed.
- Increased size of cell nuclei-the nuclei may appear to "fill" the cells.

Late
- Cessation of cell growth-cells appear to stop growing when compared to a cell-only control.
- Granular appearance
- Signs of viral budding-vesicular appearance of cells.
- Viral occlusions-few cells will contain occlusion bodies, which appear as refractive crystals in the nucleus of the insect cell.
- Detachment-cells release from the dish or flask.

Very late
- Cell lysis-a few cells may fill with occluded virus, die, and burst, leaving signs of clearing in the monolayer.

Answer Id: E9400

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I’m worried that I am not getting plaques. How many days does it take to see plaques and what size are they typically?

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Answer

Normally, very small white dots show up about 5-7 days and 1 mm plaques show up around day 10. Plaques can vary in size from 1 mm to 4 mm.

Answer Id: E9472

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

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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

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How many cells need to be infected in a 12-well plate with 1 plaque?

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Answer

Typically, 0.5 x 106 cells per well in 2.5-3 mL is a good starting point. Lysis should begin by day 3. Virus may be harvested and amplified between 3 and 7 days (90% cell death).

Answer Id: E9432

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What is the optimal time for harvesting high-titer virus following infection? What happens if I let the cells go longer?

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Answer

We recommend harvesting high-titer virus when there is 90% cell lysis. This takes approximately 5-7 days. If the cells go longer, the proteases released from the lysed cells will start to degrade viral surface proteins and result in less infectious virus.

Answer Id: E9430

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What is the thymidine kinase (TK) gene for?

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Answer

The TK gene is for negative selection of non-recombinant virus using ganciclovir.

Answer Id: E9417

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Is it necessary to include a Kozak sequence for expression of recombinant proteins in insect cells?

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Answer

While the importance of a Kozak consensus sequence in translation initiation has been demonstrated in mammalian cells, there seems to be some debate as to whether the Kozak rules are as stringent in insect cells. The only way to determine its importance would be a direct comparison of expression of the same protein from different initiation sequences. Even then, the rules for optimal expression of one protein may not hold for another. Here are two references which indicate that a Kozak consensus sequence does not have any effect on efficiency of expression in insect cells:

- Hills D, Crane-Robinson C (1995) Baculovirus expression of human basic fibroblast growth factor from a synthetic gene: role of the Kozak consensus and comparison with bacterial expression.
- Biochim Biophys Acta 1260(1):14-20.
- Ranjan A, Hasnain SE (1995) Influence of codon usage and translational initiation codon context in the AcNPV-based expression system: computer analysis using homologous and heterologous genes. Virus Genes 9(2):149-153.

Answer Id: E9406

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

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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

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What are some general suggestions for transfection in baculovirus expression systems?

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Answer

Please follow the recommendations below:

- Cells should be in excellent health, of their low passages (5-15), in log-phase growth, with viability >95%
- DNA must be of high purity, free of endotoxin
- No antibiotics should be used during transfection
- Cellfectin™ reagent has to be completely resuspended
- Include controls (media control, DNA control, and transfection reagent control) for comparison and troubleshooting

Answer Id: E9397

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