TITLE: RNAi induction
AUTHORS: Ben Jenkins

[DESCRIPTION]
Back-dilution, induction, and creating frozen E. coli feeding stocks for downstream RNAi 

[STEPS]
SECTION: Induction
6. Incubate with shaking at  
SECTION: Induction
7. Add 60 µL (0.4 millimolar (mM)) IPTG 

# This induces template expression within the L4440 plasmid construct 
SECTION: Induction
8. Incubate with shaking at  
SECTION: Match OD and freeze
10. Spin down E. coli at 3200 x g, 2 min  
SECTION: Match OD and freeze
11. Remove supernatant, and add 20 mL MQ
SECTION: Match OD and freeze
12. Spin down E. coli at 3200 x g, 2 min  

SECTION: Induction
5. In a sterile hood, add 600 µL of overnight E. coli pre-culture to 15 mL LB containing 15 µL Ampicillin and 37.5 µL Tetracycline. 

# Final concentrations Ampicillin (0.05 mg/mL), and Tetracycline (0.0125 mg/mL)
# Conduct remainder of experiment under sterile conditions
SECTION: Preparing stocks
1. In a sterile hood, streak out HT115 E. coli frozen stock onto LB Agar plate (containing 0.05 mg/mL Ampicillin and 0.0125 mg/mL Tetracycline)

# Ampicillin selects for the L4440 plasmid containing the knock-down construct
In a sterile hood, treak out HT115  E. coli strain (deficient in RNAse III)
SECTION: Preparing stocks
2. Incubate overnight at 37 °C 

SECTION: Set up
3. In a sterile hood, pick a single colony to inoculate 10 mL  LB  containing 10 µL Ampicillin and 25 µL Tetracycline. 

# Final concentrations Ampicillin (0.05 mg/mL), and Tetracycline (0.0125 mg/mL)
SECTION: Set up
4. Incubate LB overnight with shaking at   
SECTION: Set up
3.1. Streak used inoculation loop onto a fresh LB Agar plate (containing 0.05 mg/mL Ampicillin and 0.0125 mg/mL Tetracycline) to create a back-up stock plate

Incubate LB Agar plate overnight at 37 °C 
SECTION: Match OD and freeze
13. Resuspend with  3 mL NCL

# NCL (containing Ampicillin, IPTG, and β-sitosterol) prepared earlier
SECTION: Match OD and freeze
14. Add 800 µL of NCL and 200 µL resuspended E. coli to a cuvette, and measure OD600

# Use OD dilution calculator: https://docs.google.com/spreadsheets/d/1hJUsVOjwcuhKSeFQaAbNhn1ZXleTpNXN8RAR4g7zWDE/edit?usp=sharing

# Make sure to pipette E. coli up and down 3 times before removing from falcon, and again after adding to cuvette for OD measurement
SECTION: Match OD and freeze
15. In a sterile hood, add required volumes of resuspended E. coli and NCL to give 1.5 mL of E. coli  at OD 3 

# Use OD dilution calculator as above
# Larger experiments may require greater volumes, scale up your stock accordingly
# Conduct remainder of the protocol under sterile conditions

SECTION: Match OD and freeze
16. Add 37.5 µL of 80% glycerol per 1.5mL E. coli (to give 2%), and vortex

SECTION: Induction
9. In a sterile hood, prepare 150 mL NCL containing 300 µL Ampicillin,  600 µL IPTG, and 30 µL β-sitosterol

#  Ampcillin (0.1 mg/mL ), IPTG (0.4 millimolar (mM)), and  β-sitosterol (0.0008 mg/mL)
SECTION: Match OD and freeze
17. Add 60 µL of E. coli per well to a 96-well plate

# Ensure E. coli are in a consistent layout for every 96-well plate
# This is enough for 20 aliquots per E. coli stock
# Do not use the outside wells
SECTION: Match OD and freeze
18. Add lids to each stock plate, and store at -20 °C until required