How big is flashBAC?
flashBAC is approximately 136 kb in size
Which flashBAC system to use?
For simple and intracellular proteins the original flashBAC system can be used. For secreted and membrane proteins flashBACGOLD system is suited. For all of the above proteins and difficult to express proteins flashBACULTRA system is the best choice.
What is the control 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). The lacZ gene was inserted using bamH1 and Bgl2 cloning sites. This vector 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 (such as a pOET vector). The control transfer vector is named AcRP23-lazZ and more information is available in the paper R.D.Possee and S.C.Howard, Nucleic Acids Research Volume 15 Number 24, pp10233-10428 (1987)
What transfer vectors are compatible with flashBAC?
All vectors based on homologous recombination at the polh locus. We recommend the use of our pOET vectors as a first choice but flashBAC is also compatible with a wide range of transfer vectors from other suppliers.
Can I use transfer vectors pFastBac, BlueBacHis2 and pMelBac from Invitrogen with flashBAC?
Unfortunately these vectors are designed for use only with Invitrogen's Bac-n-Blue or bac-to-bac system and as such are not compatible with flashBAC. If you are using one of these vectors we do offer an upgrade service where we can transfer your genes of interest into the flashBAC system quickly and cost effectively. Please contact us for more details or download a PDF for more information.
Is it necessary to linearise the transfer vector before doing the homologous recombination reaction?
No, it works fine without being linearised.
Can I use T.ni cells for amplifying my virus?
We recommend not using T.ni cells for virus amplification because of theirpropensity for the generation of defective viruses which has been observedover many years e.g. Kumar S, Miller LK. 'Effects of serial passage ofAutographa californica nuclear polyhedrosis virus in cell culture.' VirusRes. 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 withSf9 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 oldtechnology of separating parental from recombinant virus by plaque picking,because all virus produced is recombinant. 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 proteinproduction for screening, in which case it is not necessary to know thetitre. We routinely add 0.5 ml seed stock (untitred) to 50ml cells toproduce working inoculum, it can then be titred at this stage if we need anaccurate 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 issimply 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 isusually 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 goaccording 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 baculoQUANT product page to determine highly accurate titres for fresh stocks. Alternatively visit our titration service page for our quick baculovirus titration service.
- How big is flashBAC?
- Which flashBAC system to use?
- What is the control transfer vector supplied in the flashBAC kit?
- What transfer vectors are compatible with flashBAC?
- Can I use transfer vectors pFastBac, BlueBacHis2 and pMelBac from Invitrogen with flashBAC?
- Is it necessary to linearise the transfer vector before doing the homologous recombination reaction?
- Can I use T.ni cells for amplifying my virus?
- Can I use T.ni cells for my protein production?
- Why do I not need to do a plaque assay to separate the virus?
- Do I need to do a plaque assay before I produce protein?
- Is there an easy way to titre my virus?