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Product FAQ

I'm trying to generate recombinant bacmid DNA using the Bac-to-Bac™ expression system and am seeing very poor blue/white colony differentiation. What should I do?

Answer

Poor color differentiation for your colonies could be caused by the following:

- Agar is not at the correct pH: Adjust pH of LB agar to 7.0.
- Intensity of the blue color is too weak; ensure that you are using Bluo-gal, not X-Gal. You can also try increasing the concentration of Bluo-gal to 300 μg/mL.
- Too many or too few colonies on the plate: Adjust the serial dilutions of cells to obtain an optimal number of colonies.
- Incubation period too short or temperature too low: Do not pick colonies until 48 hours after plating; incubate plates at 37 degrees C.
- IPTG concentration is not optimal: A range of 20-60 μg/mL IPTG generally gives optimal color development.

Answer Id: E9442

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Product FAQ

What is the size of the baculovirus polyhedron protein?

Answer

The polyhedron protein is 30 kDa.

Answer Id: E9408

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I am trying to isolate my bacmid DNA but the overnight culture did not grow. What could have happened?

Answer

This could be caused by the following:

- Wrong antibiotic or old media: use fresh media.
- Colonies are too old or too small: Use large white colonies from freshly streaked plates.
- Unstable insert caused by special feature of the gene of interest; for example, direct repeats: Incubate the culture at 30 degrees C for 24 hours instead of 37 degrees C overnight.

Answer Id: E9443

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Product FAQ

What is the difference between the Bac-N-Blue™ expression system and the Bac-to-Bac™ expression system?

Answer

In the Bac-N-Blue™ system, recombination between the transfer vector and the baculovirus DNA occurs in insect cells. The Bac-N-Blue™ vector is a linearized AcMNPV derivative that contains an incomplete (3’) lacZ fragment. The corresponding transfer vector contains a 5’ lacZ fragment. Upon homologous recombination, the recombinant Bac-N-Blue™ baculovirus DNA will have a complete lacZ gene that is under the control of the PETL promoter. Thus, recombinant Bac-N-Blue™ baculovirus will provide blue plaques in the plaque assay and can be easily identified. In the Bac-to-Bac™ expression system, recombination or site-specific transposition between transfer and baculovirus DNA occurs in E. coli (DH10Bac). In the Bac-to-Bac™ expression system, selection of colonies containing recombinant baculovirus DNA occurs in the presence of Luria Agar plates with 50 ™μg/mL kanamycin (bacmid), 7 ™μg/mL gentamycin (pFastBac™), 10 ™μg/mL tetracycline (helper plasmid), 100 ™μg/mL Bluo-gal, and 40 ™μg/mL IPTG.

Answer Id: E9419

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Product FAQ

I’m interested in expressing a large protein, larger than 130 kDa. Can I use the baculovirus system or will the proteins be degraded?

Answer

Our R&D team has successfully expressed proteins up to 300 kDa. If they express in >2% serum, it should minimize degradation. If you don’t mind the extra step of purification, 10% serum could be used. We highly recommend doing a time-course infection with high-titer stock, with a MOI of 5-10, to make an assessment of the minimum harvesting time necessary for the best expression. Time points should be taken every 24 hours for 5 days.

Answer Id: E9409

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Product FAQ

My bacmid DNA is degraded. What could be the cause for this and what recommendations do you have to fix this?

Answer

Please see the possible reasons and suggestions below:

- DNA stored improperly: Ensure that purified bacmid DNA is stored at -20 degrees C in aliquots to avoid repeated free/thaws.
- High molecular weight bacmid DNA handled improperly: When isolating bacmid DNA, do not vortex the DNA solution; additionally, do not resuspend DNA pellets mechanically; allow solution to sit in the tube with occasional tapping.

Answer Id: E9444

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Product FAQ

Are there preferred secretion signals for use with baculovirus?

Answer

Insect-derived signal peptides and/or prosequences cannot always enhance the expression and/or secretion of foreign secretory pathway proteins in the baculovirus system. Please see the following references:

- Jarvis DL, Summers MD, Garcia A Jr, Bohlmeyer DA (1993) Influence of different signal peptides and prosequences on expression and secretion of human tissue plasminogen activator in the baculovirus system. J Biol Chem 268(22):16754-16762.
- Tessier DC, Thomas DY, Khouri HE, Laliberte F, Vernet T (1991) Secretion of a plant protein in the baculovirus system was enhanced when its signal peptide was replaced with an insect-derived signal peptide. Gene (Amst.) 98:177-183.

Answer Id: E9410

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Product FAQ

My PCR analysis of the recombinant bacmid gives no product. What could be the cause of this and do you have any recommendations on how to fix this?

Answer

Please see the possible causes and suggestions we have to alleviate this problem:

- Use pure proofreading polymerase for analysis: Use a Taq-based polymerase for the analysis.
- Insert is very long and causes difficulties in PCR: Instead of using both M13 forward and reverse primers, use one gene-specific primer paired with the M13 primer of your choice.
- Long GC-rich stretches in the gene of interest: Consider using DMSO (up to 8%) in the PCR reaction.

Answer Id: E9445

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Product FAQ

I am purifying my secreted protein expressed in insect cells with a His-tagged purification column and getting no yield of my protein. Why and what can I do?

Answer

Media used to culture insect cells usually have an acidic pH (6.0-6.5) or contain electron-donating groups that can prevent binding of the 6xHis-tagged protein to Ni-NTA. Amino acids such as glutamine, glycine, or histidine are present at significantly higher concentrations in media for growing insect cells than in media for growing mammalian cells, and compete with the 6xHis-tagged protein for binding sites on Ni-NTA matrices. Grace’s medium (Thermo Fisher Scientific), for example, contains approximately 10 mM glutamine, 10 mM glycine, and 15 mM histidine.

Dialysis of the medium against a buffer with the appropriate composition and pH (8.0) similar to the lysis buffer recommended for purification under native conditions usually restores optimal binding conditions. Note that depending on the medium used, a white precipitate (probably made up of insoluble salts) can occur, but normally the 6xHis-tagged protein remains in solution. This can be tested by either protein quantitation if using a protein-free medium or by monitoring the amount of 6xHis-tagged protein by western-blot analysis. After centrifugation, 6xHis-tagged protein can be directly purified from the cleared supernatant.

Answer Id: E9456

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Product FAQ

I'm trying to generate recombinant bacmid DNA using the Bac-to-Bac™ expression system and all the colonies obtained are white. Shouldn't I expect to see some blue colonies?

Answer

Although you will be picking white (recombinant) colonies, you should expect to see some blue (contain non-recombinant bacmid) colonies. Here are some possible causes for seeing no blue colonies and recommendations for the same:

- Insufficient time for color development: Wait at least 48 hours before identifying colony phenotypes.
- Use Bluo-gal instead of X-Gal in agar plates: Use Bluo-gal in plates to increase contrast between blue and white colonies.
- Insufficient growth after transposition: Grow transformed cells in SOC medium for a minimum of 4 hours before plating.
- Bluo-gal and IPTG omitted from plates: Prepare fresh selective plates containing 50 μg/mL kanamycin, 7 μg/mL gentamicin, 10 μg/mL tetracycline, 100 μg/mL Bluo-gal, and 40 μg/mL IPTG.
- There are too many colonies on the plate: Serially dilute the transformation mix to obtain well-spaced colonies (10-2 to 10-4 is suggested).
- Plates are too old or stored in light: Do not use plates that are more than 4 weeks old; store plates protected from light.
- Incubation period too short or temperature is too low: Wait at least 48 hours before picking colonies. Incubate plates at 37 degrees C.

Answer Id: E9438

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Product FAQ

Is there a packaging limit for the baculovirus?

Answer

The baculovirus rod will continue to elongate as required to package the DNA. Thus, the system could theoretically accommodate hundreds of Kb. Standard cloning techniques will limit the insert size before packaging limits become an issue.

Answer Id: E9411

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Product FAQ

My bacmid DNA contains a mixture of recombinant bacmid and empty bacmid. What am I doing wrong?

Answer

Most likely, a colony that was gray or dark in the center was picked. Try to analyze more white DH10Bac™ transformants. Typically, we recommend picking a white colony whose diameter is >2 mm. Restreak the white colonies on a fresh plate with 50 μg/mL kanamycin, 7 μg/mL gentamicin, 10 μg/mL tetracycline, 100 μg/mL Bluo-gal and 40 μg/mL IPTG. Incubate plates for 24 hours.

Answer Id: E9446

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Product FAQ

Both transfected cells and Cellfectin™ II Reagent alone negative control cells are dead within 36 hours. What could cause this?

Answer

There are several possibilities:

- Using media containing antibiotics during transfections.
- Plating cells at too low a density: We recommend at least 70% confluence.
- Using cells at too early a passage: We recommend growing cells for at least 5 passages before using them for transfection.
- Contamination because of no pen/strep after the transfection: After 5-8 hr incubation with the transfection mixture, remove the mixture and add antibiotics containing media/well.

Answer Id: E9448

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Product FAQ

I'm trying to generate recombinant bacmid DNA using the Bac-to-Bac™ expression system and all of my colonies are blue. What could be the cause of this and what should I do?

Answer

Please review the following possibilities and recommendations:

- pFastBac™ DNA used for transformation was of poor quality: Use purified plasmid DNA for transformation and check the quality of your plasmid DNA.
- Gentamicin omitted from plates: Prepare fresh selective plates containing 50 μg/mL kanamycin, 7 μg/mL gentamicin, 10 μg/mL tetracycline, 100 μg/mL Bluo-gal, and 40 μg/mL IPTG.

Answer Id: E9439

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Product FAQ

I don’t see any signs of cell infection 72 hours after infection with my P2 viral stock. Why is this?

Answer

Check the MOI. It may be low because the titer of the P1 virus is lower than what was estimated.

Answer Id: E9450

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