flashBAC is approximately 136 kb in size,

What is the transfer vector supplied in the flashBAC kit?

The transfer vector supplied with the kit contains a marker gene (lacz) under control of the polyhedrin gene (polh) and is supplied as a positive control only, i.e. to confirm that recombination within the insect cells has occurred and the virus is expressing. This would be done alongside your own transfer vector containing your gene of interest.

What vectors are compatible with flashBAC?

All vectors based on homologous recombination at the polh locus (see list)

Can I use transfer vectors BlueBacHis2 and pMelBac from Invitrogen with flashBAC?

Unfortunately these vectors are designed for use only with Invitrogens Bac-n-Blue system and as such are not compatible with flashBAC.

Is it necessary to linearize the transfer vector before doing the homologous recombination reaction?

No.

Can I use T.ni cells for amplifying my virus?

We recommend not using T.ni cells for virus amplification because of their propensity for the generation of defective viruses which has been observed over many yearse.g. Kumar S, Miller LK. ‘Effects of serial passage of Autographa californica nuclear polyhedrosis virus in cell culture.’ Virus Res. 1987 Jun;7(4):335-49.The protocol we prefer to use is the one contained within the flashBAC manual, using Sf9 cells for co-transfections and amplifications and Sf21 cells for plaque assays.

Can I use T.ni cells for my protein production?

Yes. T.ni cells can give excellent protein production results and can be used for this purpose without problems. We would recommend a comparison with Sf9 cells to evaluate optimal protein production for your specific protein.

Why do I not need to do a plaque assay to separate the virus?

In the initial stages a plaque assay is not required - ie there is no need to go through the stage that is necessary in systems based on the old technology of separating parental from recombinant virus by plaque picking, because all virus produced is recombinant (http://www.expressiontechnologies.com/flashBAC/technology.asp) It's not possible to know an exact moi without doing a plaque assay at the end, but often all that is required is a sample of the virus for a quick protein production for screening, in which case it is not necessary to know the titre. We routinely add 0.5 ml seed stock (untitred) to 200ml cells to produce working inoculum, it can then be titred at this stage if we need an accurate moi, but the titre is usually in the high 10^7 / 10^8 pfu/ml.

Do I need to do a plaque assay before I produce protein?

This depends on whether you just want a protein sample for screening purposes or if you want to produce optimal yield of protein. If your aim is simply to screen, it is not necessary to know the titre. Simply use 100 – 200ul to infect cells in a 35mm dish, or 400 – 500 ul to infect cells in a T25 flask. For larger quantities of inoculm, 0.5 ml untitred seed stock can be added to 200ml cells to produce a working inoculum. If required, this can then be titred to give an accurate m.o.i. for infection, but the titre is usually in the region of high 10^7 / 10^8.The problem with not titrating working stocks of virus is that sometimes virus amplification does not go according to plan (often due to the cell culture – see above) and the final titre can be much lower than 10^7 pfu/ml. If this happens there is unlikely to be good protein expression.

Is there an easy way to titre my virus?

Visit the BaculoQuantTM page of our website for our quick baculovirus titration service