How do you make Bacmid?
How do you make Bacmid?
3.1. 1 Bacmid DNA preparation
- Mix 10 ng of DNA with 50 μL of DH10Bac E.
- Incubate on ice for 30 min.
- Heat shock at 42 °C for 45 s.
- Incubate on ice for 2 min.
- Add 400 μL SOC media to the tube.
- Recover cells in a shaking incubator at 37 °C for 4 h.
- Plate 200 μL of cells on a bacmid plate.
- Incubate at 37 °C for 48 h.
How big is a Bacmid?
Notes: Recombinant bacmid is >135 kb in size. Since restriction analysis is difficult to perform with DNA of this size, PCR analysis is recommended to verify the presence of your gene of interest in the recombinant bacmid.
Is a Bacmid a plasmid?
The bacmid is essentially a very large plasmid containing the baculovirus genome modified to carry a lacZ gene and an attTn7 docking site inserted in the lacZ coding region. The helper plasmid expresses the Tn7 transposase.
Can baculovirus infect human cells?
Interestingly, baculovirus can infect mammalian cells very efficiently, although it replicates only in insect cells.
What are Sf9 cells used for?
Sf9 cells, a clonal isolate of Spodoptera frugiperda Sf21 cells (IPLB-Sf21-AE), are commonly used in insect cell culture for recombinant protein production using baculovirus. They were originally established from ovarian tissue. They can be grown in the absence of serum, and can be cultured attached or in suspension.
How do you amplify baculovirus?
The Quick Amplification Guide suggests the best way to amplify, should the baculovirus yield be lower than average. Add 2ml PI stock to 50ml Sf9 cells at 2×106 cells/ml in a 1L Erlenmeyer flask. Add 2ml PI stock to 10ml Sf9 cells at 2×106 cells/ml in a 125ml Erlenmeyer flask.
What is the size of baculovirus?
Baculoviruses are a very diverse group of viruses with double-stranded, circular, supercoiled genomes, with sizes varying from about 80 to over 180 kb, that encode between 90 and 180 genes.
What does baculovirus mean?
Baculoviruses, which belong to the family Baculoviridae, are a group of DNA viruses that infect insects. These viruses are very small, rod-shaped structures, composing of a protein coat containing a small amount of DNA which encodes components for further viral reproduction.
Is baculovirus a biocontrol agent?
The majority of baculoviruses used as biological control agents are in the genus Nucleopolyhedrovirus, so “baculovirus” or “virus” will hereafter refer to nucleopolyhedroviruses. These viruses are excellent candidates for species-specific, narrow spectrum insecticidal applications.
How does baculovirus expression system work?
The BaculoDirect baculovirus expression system (Invitrogen) uses the Gateway technology to permit the direct transfer of a gene of interest into the baculovirus genome, without the need for production of recombinant bacmid DNA.