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

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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

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

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

Answer Id: E9467

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My agarose overlay is floating. Is this because I did not aspirate the medium completely?

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Answer

Yes, this is indicative of an aspirating problem on the plaques. The agarose overlays were “floating” because the medium was not completely aspirated from the plates. The plates need to be completely dry before the agarose is placed over the cells, especially when plaques will be picked. To do this, we typically tip the plate slightly and keep going around the rim of the plate with the Pasteur pipette tip, being careful not to disturb the cell monolayer. If any medium pooling at the rims of the plates (they will be small pools) is seen, continue to aspirate. This “floating” agarose overlay problem may also result in wild-type contamination. The wild-type virus is able to migrate to other portions of the plates and contaminate recombinant plaques. Wild-type virus replicates much faster than recombinant virus, and can quickly overwhelm the recombinant virus.

Answer Id: E9468

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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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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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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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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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What primer purity should be used for adding attB sites to my PCR product?

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Answer

Standard desalted purity is generally sufficient for creating attB primers. We examined HPLC-purified oligos for Gateway™ cloning (about 50bp long) and found only about a 2-fold increase in colony number over standard desalted primers. If too few colonies are obtained, you may try to increase the amount of PCR product used and/or incubate the BP reaction overnight.

Answer Id: E3172

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

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

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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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What is the multiplicity of infection, and how can I calculate it?

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The MOI, or multiplicity of infection, is the average number of viral particles that infect a single cell in a specific experiment. You can calculate the MOI with the following equation:
MOI (pfu/cell) = [titer (pfu) x viral stock volume (mL) used in inocula] / [cell density (cells/mL) x culture volume (mL)]

Answer Id: E9404

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From where does Gateway™ get its lambda nomenclature, and is it consistent with textbook nomenclature for lambda recombination?

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Answer

The Gateway™ nomenclature is consistent with lambda nomenclature, but we use numbers to differentiate between modified versions of the att sites (attB1, attB2, attP1, attP2, and so on). We have introduced mutations in the att sites to provide specificity and directionality to the recombination reaction. For example, attB1 will only recombine with attP1 and not with attP2.

Answer Id: E3209

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What is the difference between LR Clonase™ II and LR Clonase™ II Plus?

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Answer

LR Clonase™ II Plus contains an optimized formulation of recombination enzymes for use in MultiSite Gateway™ LR reactions™. LR Clonase™ and LR Clonase™ II enzyme mixes are not recommended for MultiSite Gateway™ LR recombination reactions, but LR Clonase™ II Plus is compatible with both multi-site and single-site LR recombination reactions.

Answer Id: E5237

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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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Must PCR conditions be changed once the original PCR primers have attB sequence added to them?

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Answer

Since the attB sequences are on the 5' end of oligos, they will not anneal to the target template in the first round of PCR. Sometimes the PCR product is more specific with the attB primers, probably due to the longer annealing sequence (all of attB plus gene specific sequence) after the first round of amplification. Generally there is no need to change PCR reaction conditions when primers have the additional attB sequence

Answer Id: E3222

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