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From 2012.igem.org

Contents 1 Protocol 1.1 Medium & Buffer recipes 1.2 CCMV Coat Protein VLP formation 1.3 Hepatitis B Coat Protein VLP formation 1.4 Procedure 1.5 TuYV Coat Protein VLP formation 1.6 PLRV Coat Protein VLP formation 1.7 General Protocol

Protocol

Medium & Buffer recipes

• LB medium
• SOB medium
• SOC medium
• Disassembly buffer
• Dialysis buffer 10x
• Reassembly buffer
• Virus buffer
• Towbin's electrotransfer buffer 1x
• Towbin's electrotransfer buffer 10x

CCMV Coat Protein VLP formation

• Growing culture
• Dialysis of the VLPs
• Purifing the VLPs

Hepatitis B Coat Protein VLP formation

• Growing culture
• Dialysis of the VLPs
• Purifing the VLPs

Procedure

1. Prepare a Erlenmeyer flask with 50mL of LB-medium and keep it at 37°C
2. Pick E.coli BL21 from a plate and grow them over night in a 10 mL LB culture containing 50mg/L Kanamycin at 37°C
3. Inoculate the large flask with 1 mL of the overnight culture and grow at 37°C for 2.5h until the OD600 reaches approx. 0.6
4. Induce with 1.25 mM IPTG (0.25 mL of a 250 mM stock)
5. Incubate at 37°C for 4 h
6. Centrifuge the cells in 50ml greiner tubes at 4700 rpm for 18 minutes
7. Decant the supernatant and resuspend the pellets in ± 10 mL washing buffer each
8. Centrifuge again at 4700 rpm for 18 minutes
9. Decant the supernatant and centrifuge for another minute
10. Pipette any liquid to clear the tube of supernatant
11. (possible to freeze the cells to -20°C at this point)

TuYV Coat Protein VLP formation

PLRV Coat Protein VLP formation

• RNA isolation from potato leaf material

General Protocol

• Preparation of electrocompetent cells of E. coli
• SDS-Polyacrylamide and native gel electrophoresis
• Dynamic Light Scattering user manual
• French press user manual
• FPLC user manual