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Product FAQ

I’m getting a low-titer P1 viral stock and would like to generate a high-titer stock. What should I do?

Answer

To get a high-titer stock, reinfect cells with the P1 stock and generate a P2 high-titer stock. Follow the directions in the BaculoDirect™ manual on page 18 to generate your P2 stock.

Answer Id: E9460

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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

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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Product FAQ

I'm trying to generate recombinant bacmid DNA using the Bac-to-Bac™ expression system and I’m getting very few colonies. What could be the cause for this and do you have any recommendations for how to fix this?

Answer

Please review the following reasons and our recommendations:

- Use LB medium for recovery/expression period: Use SOC medium for the 4 hr growth time.
- Recovery/expression time too short: Increase the recovery time to > 4 hr at 37 degrees C or 6 hr at 30 degrees C.
- IPTG concentration is not optimal: We suggest using 20-40 μg/mL IPTG.

Answer Id: E9440

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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 cannot detect any recombinant fusion protein after using the BaculoDirect™ Expression Kit. What could be the cause for this and what do you suggest I try?

Answer

Please check the construction of your entry clone, and ensure that the insert is in frame with the vector. Analyze the recombinant viral DNA by PCR to confirm the correct size and orientation of your insert after the LR reaction. Sequence your PCR product to verify the proper reading frame for expression of the epitope tag.

Answer Id: E9461

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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

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

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

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

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

You offer competent cells in Subcloning Efficiency™, Library Efficiency™ and MAX Efficiency™. How do these differ?

Answer

There are a few exceptions, but in general the difference is in guaranteed transformation efficiency as follows:
Subcloning Efficiency™ cells are guaranteed to produce at least 1.0 x 10E6 transformants per µg of transformed pUC19 or pUC18 supercoiled plasmid
Library Efficiency™ cells are guaranteed to produce at least 1.0 x 10E8 transformants per µg pUC19 or pUC18 DNA
MAX Efficiency™ cells are guaranteed to produce at least 1.0 x 10E9 transformants per µg pUC19 or pUC18 DNA

Answer Id: E3869

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Product FAQ

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

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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Product FAQ

When should DMSO, formamide, glycerol and other cosolvents be used in PCR?

Answer

Cosolvents may be used when there is a failure of amplification, either because the template contains stable hairpin-loops or the region of amplification is GC-rich. Keep in mind that all of these cosolvents have the effect of lowering enzyme activity, which will decrease amplification yield. For more information see P Landre et al (1995). The use of co-solvents to enhance amplification by the polymerase chain reaction. In: PCR Strategies, edited by MA Innis, DH Gelfand, JJ Sninsky. Academic Press, San Diego, CA, pp. 3-16.

Additionally, when amplifying very long PCR fragments (greater than 5 kb) the use of cosolvents is often recommended to help compensate for the increased melting temperature of these fragments.

Answer Id: E1320

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Product FAQ

I performed an LR reaction, followed by transfection into Sf9/Sf21 insect cells, but do not see any signs of infection even though it’s been 72 hours. What should i do?

Answer

Please see our recommendations below:

- Check the LR reaction by PCR analysis prior to transfection into insect cells.
- We recommend using Grace’s Insect Cell Culture Medium, Unsupplemented during the transfection experiment instead of serum-free medium, as components in serum-free medium may interfere with transfection.
- Ensure that FBS, supplements, or antibiotics are not included during transfection, as the proteins in these materials can interfere with the Cellfectin™ II Reagent.
- Use the LR recombination reaction using the pENTR/CAT plasmid as a positive control and Cellfectin™ II Reagent only (mock transfection) as a negative control.
- Ensure that cells are in the log phase of growth with >95% viability, and the amount of cells are in accordance with the suggestions in the manual.
- Cells may not show signs of viral infection for up to a week depending on transfection efficiency; continue culturing and monitor cells daily for signs of infection.

Answer Id: E9459

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