Invitrogen™ MAX Efficiency™ DH10Bac Competent Cells

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.

Low protein yield may occur due to the following reasons:

- Viral stock contains a mixture of recombinant and non-recombinant baculovirus: Perform plaque purification to isolate recombinant baculovirus.
- Baculovirus is not recombinant: Verify transposition by PCR analysis of bacmid DNA using pUC/M13 forward and reverse primers; re-transfect insect cells with new recombinant bacmid DNA.
- Use too low or too high viral titer: Vary the MOI.
- Time of cell harvest is not optimal: perform a time course of expression to determine the optimal time to obtain maximal protein expression.
- Cell growth conditions and medium are not optimal: Optimize cell culture conditions based on the size of your culture vessel and expression conditions; we recommend using Sf-900 II SFM or Sf-900 III SFM for optimal cell growth and protein expression.
- Cell line is not optimal; try other insect cell lines.
- Cells were harvested too late: Do a time-course experiment and harvest cells at different time points.

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

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.

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.

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.

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.

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

- 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.

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.

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.

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.

We recommend running 1/8th of the 40 µL midiprep sample on a 0.5% TAE agarose gel. Electrophorese slowly at 23 V for 12 hr. The banding pattern of the recombinant bacmid midiprep should be seen.

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.

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.

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.

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.

The following is an excellent reference for how to prevent proteolytic artifacts in the baculovirus expression system:
Hom LG, Volkman LE (1998) Preventing proteolytic artifacts in the Baculovirus expression system. BioTechniques 25:18-20.

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.

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.

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.

The polyhedron protein is 30 kDa.

The promoter that drives the gene of interest is the polyhedron promoter.

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.

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

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.