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Product FAQ

Can I sequence a peptide that is acetylated or biotinylated?

Answer

No. These groups effectively block the N terminus.

Answer Id: E1267

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Product FAQ

I performed my PCR and see different banding patterns. What do the different PCR sizes mean?

Answer

The expected PCR size will depend on the pFastBac™ vector that got transposed into the bacmid:

- Bacmid alone: 300 bp
- With pFastBac™ 1: 2300 bp + size of insert
- With pFastBac™ 1-Gus: 4200 bp
- With pFastBac™ HT: 2430 bp + size of insert
- With pFastBac™ HT-CAT: 3075 bp
- With pFastBac™ Dual: 2560 bp + size of insert
- With pFastBac™ Dual-Gus/CAT: 5340 bp
- With pDEST8: 2316 bp + size of insert
- With pDEST10: 2484 bp + size of insert
- With pDEST20: 3031 bp + size of insert

Answer Id: E9414

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Product FAQ

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

Answer

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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Product FAQ

How clean must my DNA be to use in a Gateway™ cloning reaction?

Answer

Mini-prep (alkaline lysis) DNA preparations work well in Gateway™ cloning reactions. It is important that the procedure remove contaminating RNA for accurate quantification. Plasmid DNA purified with our S.N.A.P.™ nucleic acid purification kits, ChargeSwitch™ kits, or PureLink™ kits are recommended.

Answer Id: E3204

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Product FAQ

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

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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Product FAQ

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

Answer

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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Product FAQ

Is it possible to co-infect insect cells with two different recombinant viruses in order to co-express, for example, two subunits of a protein?

Answer

Yes, it is possible. Several five-subunit proteins, such as human replication factor C, have been expressed using recombinant baculovirus. We recommend that a separate high-titer stock (HTS) of each subunit be produced to optimally express the multi-subunit protein. This way, the amount of each subunit expressed can be controlled by varying the multiplicity of infection (MOI) of each subunit's HTS. Please refer to the following articles for more information:

- Chen W and Bhal OP (1991) Recombinant carbohydrate and selnomethionyl variants of human choriogonadotropin. J Biol Chem 266(13):8192-8197.
- Chen WY and Bhal OP (1991) Selenomethionyl analog of recombinant human choriogonadotropin. J Biol Chem 266(15):9355-9358.
- Fabian JR, Kimball SR, Jefferson LS (1998) Reconstitution and purification of eukaryotic initiation factor 2B (eIF2B) expressed in Sf21 insect cells. Protein Expr Purif 13(1):16-22.

Answer Id: E9405

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Product FAQ

What is Gateway™ Cloning Technology?

Answer

Gateway™ Cloning Technology is an easy-to-use system for cloning and subcloning DNA segments (e.g. genes of interest), facilitating gene functional analysis, protein expression, and the integration of technology platforms. One can also readily clone PCR products into so-called Gateway™ "Entry" vectors. To shuttle inserts from one vector to another, the Gateway™ Cloning Technology uses bacteriophage lambda-based site-specific recombination. There is no need to use restriction enzymes and ligase to subclone inserts.

One advantage of Gateway™ Cloning Technology is that genes present in a single Gateway™ Entry vector can be subcloned into multiple different Gateway™ Destination vectors. After this 1 hour in vitro subcloning reaction, a high percentage of the colonies obtained carry the desired expression clone. For more details, please see the product manual for cat# 12535029 or cat# 12535037.

Answer Id: E3029

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Product FAQ

How many times can I thaw BP Clonase™ II and LR Clonase™ II?

Answer

BP Clonase™ II and LR Clonase™ II can be freeze/thawed at least 10 times without significant loss of activity. However, you may still want to aliquot the enzymes to keep freeze/thaw variability to a minimum.

These enzymes are more stable than the original BP and LR Clonase™ and can be stored at -20°C for 6 months.

Answer Id: E3205

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Product FAQ

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

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

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Product FAQ

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

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

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Product FAQ

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

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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Product FAQ

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

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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Product FAQ

What viral titer do you suggest having to infect my cells?

Answer

We suggest using a viral stock with a titer of >1 x 10e8 pfu/mL for expression studies.

Answer Id: E9426

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Product FAQ

Can I purchase the 5X LR Clonase™ buffer or 5X BP Clonase™ buffer separately?

Answer

We do not offer the 5X LR Clonase™ buffer and 5X BP Clonase™ buffer as standalone products. They are available as part of the enzyme kits.

Answer Id: E9855

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