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

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

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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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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?

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

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

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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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My transfected cells are floating up and look dead, but the Cellfectin™ II Reagent alone control plate is fine. What could be happening?

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Answer

This may be due to contamination or cytotoxicity from the bacmid prep. Make sure to include a negative control that is the bacmid only without Cellfectin™ II Reagent. Additionally, use the PureLink™ HiPure plasmid prep kit, not the silica-based miniprep kit for bacmid prep.

Answer Id: E9447

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

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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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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?

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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 X-Gal instead of Bluo-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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My viral stock used to work well from my Bac-to-Bac™ baculovirus expression system, but after a couple of months, there is much less protein expression. Why is this?

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Answer

Please review the following possibilities and solutions:

- Viral stock was amplified using high MOI originally: Go back to the lower-passage viral stock and do a low-MOI amplification.
- Did not spin down and get rid of cells when harvesting viral supernatant: Go back to the lower-passage viral stock and do a low-MOI amplification; if this viral stock is P2, this stock can be used in amplification.
- For some genes, the virus can become very unstable: Free the aliquoted P2 viral stock and do one run of amplification after reviving.

Answer Id: E9455

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How do you recommend that I prepare my DNA for successful electroporation of E. coli?

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Answer

For best results, DNA used in electroporation must have a very low ionic strength and a high resistance. A high-salt DNA sample may be purified by either ethanol precipitation or dialysis.

The following suggested protocols are for ligation reactions of 20ul. The volumes may be adjusted to suit the amount being prepared.

Purifying DNA by Precipitation: Add 5 to 10 ug of tRNA to a 20ul ligation reaction. Adjust the solution to 2.5 M in ammonium acetate using a 7.5 M ammonium acetate stock solution. Mix well. Add two volumes of 100 % ethanol. Centrifuge at 12,000 x g for 15 min at 4C. Remove the supernatant with a micropipet. Wash the pellet with 60ul of 70% ethanol. Centrifuge at 12,000 x g for 15 min at room temperature. Remove the supernatant with a micropipet. Air dry the pellet. Resuspend the DNA in 0.5X TE buffer [5 mM Tris-HCl, 0.5 mM EDTA (pH 7.5)] to a concentration of 10 ng/ul of DNA. Use 1 ul per transformation of 20 ul of cell suspension.

Purifying DNA by Microdialysis: Float a Millipore filter, type VS 0.025 um, on a pool of 0.5X TE buffer (or 10% glycerol) in a small plastic container. Place 20ul of the DNA solution as a drop on top of the filter. Incubate at room temperature for several hours. Withdraw the DNA drop from the filter and place it in a polypropylene microcentrifuge tube. Use 1ul of this DNA for each electrotransformation reaction.

Answer Id: E4159

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Is there a packaging limit for the baculovirus?

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

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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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Using the Bac-to-Bac™ baculovirus expression system, I’ve transduced my cells but do not see any signs of viral production. It has been 5 days. What happened and what should I do?

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Answer

Please see the possible reasons and suggestions below:

- Mixture of Cellfectin™ II Reagent and bacmid was not performed or was not incubated long enough: Mix the Cellfectin™ II Reagent and bacmid well by tapping or gentle vortexing, and incubate the mixture for 15-45 min.
- Bacmid yield is lower than estimated: Set up an optimization with different amounts of bacmid.
- Bacmid is sheared during purification or freeze/thaw: Verify the integrity of bacmid on a gel.
- Incubation time is not long enough: Incubate mix for 8 hr at 27 degrees C.
- Cells used are of high passages or have passed log-phase growth: For best results, use cells between 8-15 passages; plate cells when they are in log-phase growth.
- Cellfectin™ II Reagent has been frozen: Purchase a new vial.
- Medium used contains serum: Use unsupplemented Grace’s medium in transfection.

Answer Id: E9449

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What is the promoter used in the Bac-to-Bac™ expression system?

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Answer

The promoter that drives the gene of interest is the polyhedron promoter. This promoter can be substituted by the p10 promoter, though the polyhedron promoter is generally 3-5 times stronger for most proteins. However, a protein that is highly modified or secreted is often expressed much more efficiently by the p10 promoter, as it becomes active in very early late phase, as opposed to the polyhedron promoter, which is not active until very late phase. Cellular protein synthesis that is required for the efficient and correct processing of complex proteins is shut down during the very late phase. This explains why some reports mention the expression of secreted and modified proteins where the p10 promoter is just as efficient as the polyhedron promoter or as much as 2x higher than the polyhedron promoter. In most cases, however, the polyhedron promoter is working just fine. The determination as to which is the stronger promoter will depend on a number of factors, including the nature of the protein and the time of harvest post-infection.

Answer Id: E9407

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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?

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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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