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Protocols

Bacillus subtilis transformation protocol

Components of media

SpC Medium

Add the following to 10ml of TBase

• 100 ul of 50% glucose
• 150 ul of 1.2% MgSO4.3H2O solution
• 200 ul of 10% yeast extract
• 250 ul of 1% CAS amino acids

SpII medium

Add the following to 10ml of TBase

• 100 ul of 50% glucose
• 700 ul of 1.2% MgSO4.3H2O solution
• 100 ul of 10% yeast extract
• 100 ul of 1% CAS amino acids
• 50 ul of 0. 1M CaCl2

TBase

• Dissolve the following in 1L of water and autoclave
• (NH4)2SO4 - 2g
• K2HPO4.3H2O - 18.3g
• KH2PO4 - 6g
• Trisodium citrate.2H2O - 1g

Procedure

1. Streak a large patch of Bacillus subtilis cells onto an LB agar plate (2.5% LB and 1% agar)
2. Incubate at 37 deg C overnight
3. Scrape cell’s off the patch into 10 ml of freshly made SpC medium (for contents of SpC medium see below) till OD of tube becomes approximately 0.5
4. Incubate at 37 deg C and 220 rpm for 5 hours
5. Inoculate 200ul of this culture into 10 ml of freshly prepared SpII medium (for contents of SpII medium see below)
6. Incubate at 37 deg C and 170 rpm for 90 min
7. Pellet cells in 1.5 ml microcentrifuge tubes. Decent and store supernatant
8. Resuspend pellets in 200ul of supernatant
9. Add 15ul of plasmid (pBS1C backbone)
10. Incubate at 37 deg C
11. Plate on LB agar plates (2.5% LB and 1% agar) containing chloramphenicol (25 ug/ml)
12. Incubate overnight at 37 deg C to grow colonies

Plasmid Preparation

GeneAll ExprepTM plasmid SV mini protocol used. Link provided below for the same:
GeneAll Prep

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