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.
What is Bac to Bac system?
The Bac-to-Bac baculovirus expression system enables the efficient production of recombinant baculovirus for expression testing in insect cells. The system relies on generation of recombinant baculovirus by site-specific transposition in E. coli rather than homologous recombination in insect cells.
Which baculovirus are used as biological control agents?
UPLOAD PHOTO AND GET THE ANSWER NOW! Solution : Baculoviruses are used as biological control agentsthat attack insects and other artropods….Baculoviruses are used as biological control agents that attacks.
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What is Sf9 insect cell?
The Sf9 insect cell line is a clonal isolate derived from the parental Spodoptera frugiperda cell line IPLB-Sf-21-AE, and it is a suitable host for expression of recombinant proteins from baculovirus expression systems (e.g., Invitrogen’s Bac-to-Bac® and Bac-N-Blue™ Expression Systems).
Are Sf9 cells immortalized?
Of the immortalized insect cells available, the most commonly utilized lines are derived from Spodoptera frugiperda (fall armyworm) with Sf9 and Sf21 being the most common.
What is the Bacmid?
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.
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.
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.