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Product FAQ

What should I look for to indicate a successful transfection in which baculovirus is produced?

Answer

Adherent Sf9 cells round up and show a smaller contact point. Infected Sf9 cells in suspension culture round up and look larger when infected.

Answer Id: E9398

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

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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Product FAQ

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

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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Product FAQ

Do I need to purify my recombinant virus away from an uncut or non-recombinant viral DNA?

Answer

Yes. Contamination of your recombinant DNA with uncut (occ+) DNA will lead to dilution of your recombinant virus over time because, in general, uncut (wild-type, occ+) virus infects and replicates at higher efficiency than recombinant virus. Also, initiating expression studies with a pure, single virus population will ensure reproducible results.

Answer Id: E9401

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Product FAQ

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

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

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Product FAQ

How can I store my viral stock?

Answer

If the medium is serum-free, add serum to 10%. Serum proteins act as substrates for proteases and therefore prevent degradation of viral coat proteins. Store viral stocks at 4 degrees C, and protect from light. Aliquots can be stored at -80 degrees C, but viral titer should be checked before use, as freeze/thaw cycles of the virus can result in a 10- to 100-fold decrease in viral titer.

Answer Id: E9402

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Product FAQ

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

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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Product FAQ

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

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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Product FAQ

Can I scale-up the production of recombinant protein using the baculovirus expression system? If so, what methods are available?

Answer

Yes, large-scale expression experiments can be performed. Please see below for different large-scale methods, requirements, added benefits, and references:
- Stirred bioreactor
- Airlift fermentor
- Insect larvae

Answer Id: E9403

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Product FAQ

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

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

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Product FAQ

I’d like to perform PCR on a plaque, and also make a stock from the same plaque. How do you suggest I do this?

Answer

Our R&D team will typically pick a plug and add it to a 12-well dish with 0.5 x 10e6 cells/well and 2.5 mL total volume per well. After approximately 3 days, remove 0.75 mL to make DNA for PCR and keep the remaining medium in an Eppendorf tube as your P1 viral stock. As an aside, it is okay to pick a plaque and store it in Grace’s medium.

Answer Id: E9431

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Product FAQ

What do attL1 and attL2 sites look like after recombination between attB and attP sites?

Answer

The attP1 sequence (pDONR™) is:
AATAATGATT TTATTTTGAC TGATAGTGAC CTGTTCGTTG CAACAAATTG ATGAGCAATGCTTTTTTAT AATGCCAACT TTGTACAAAA AAGC[TGAACG AGAAACGTAA AATGATATAA ATATCAATAT ATTAAATTAG ATTTTGCATA AAAAACAGACTA CATAATACTG TAAAACACAA CATATCCAGT CACTATGAAT CAACTACTTA GATGGTATTA GTGACCTGTA]

The region within brackets is where the site is "cut" and replaced by the attB1-fragment sequence to make an attL1 site. The sequence GTACAAA is the overlap sequence present in all att1 sites and is always "cut" right before the first G.

The overlap sequence in attP2 sites is CTTGTAC and cut before C. This is attP2:
ACAGGTCACT AATACCATCT AAGTAGTTGA TTCATAGTGA CTGGATATGT TGTGTTTTAC AGTATTATGT AGTCTGTTTT TTATGCAAAA TCTAATTTAA TATATTGATA TTTATATCAT TTTACGTTTC TCGTTCAGCT TTCTTGTACA AAGTTGGCAT TATAAGAAAG CATTGCTTAT AATTTGTTG CAACGAACAG GTCACTATCA GTCAAAATAA AATCATTATT

So, attL1 (Entry Clone) should be:
A ATAATGATTT TATTTTGACT GATAGTGACC TGTTCGTTGC AACAAATTGA TGAGCAATGC TTTTTTATAA TGCCAACT TT G TAC AAA AAA GC[A GGC T]NN NNN

attL2 (Entry Clone) should be:
NNN N[AC C]CA GCT TT CTTGTACA AAGTTGGCAT TATAAGAAAG CATTGCTTAT CAATTTGTTG CAACGAACAG GTCACTATCA GTCAAAATAA AATCATTATT

The sequence in brackets comes from attB, and N is your gene-specific sequence.

Note: When creating an Entry Clone through the BP reaction and a PCR product, the vector backbone is not the same as Gateway™ Entry vectors. The backbone in the case of PCR BP cloning is pDONR™201.

Answer Id: E3225

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Product FAQ

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

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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Product FAQ

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

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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Product FAQ

Do you offer Gateway™ vectors for expression in plants?

Answer

We do not offer any Gateway™ vectors for expression in plants.

Answer Id: E9854

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