0.1M CaCl2-MgCl2 (20mM CaCl2 and 80mM MgCl2) Solution
0.1M CaCl2-15% Glycerol Solution
Procedures
Strain to be prepared competent is streaked on LB agar plate and cultured overnight.
Pick single colony and culture in 2ml LB medium overnight.
Add 1ml overnight culture to 100ml LB medium.
Incubate culture for about 3h, until OD600 reading reaches 0.4.
Transfer culture into 50ml Falcon tube and chill on ice for 10min.
Centrifuge chilled culture at 4000rpm, 4°C for 10min.
Discard supernatant and resuspend using ice-chilled 0.1M CaCl2-MgCl2 solution 30ml. Place on ice for 30min.
Centrifuge culture at 4000rpm, 4°C for 5min.
Discard supernatant and resuspend using ice-chilled 0.1M CaCl2-15% glycerol solution.
Distribute the resuspended solution to 1.5ml centrifuge tube at 200ul per tube.
Store cell at -80°C.
Miniprep
Material
Axygen Miniprep Kit
1.5mL Eppendorf tubes
Procedures
Centrifuge overnight cell culture at 4700rpm for 6min.
Discard supernatant.
Add 200ul FAPD1 buffer to Falcon tube and resuspend cell pellet.
Transfer all liquid to labelled centrifuge tube.
Add 200ul FAPD2 buffer and gently invert tube for 10 times. DON'T vortex!
Add FAPD3 buffer and invert tube 10 times immediately.
Centrifuge at full speed for 5min.
Pour supernatant into filter column, spin down at full speed for 30s. In case of low expected DNA yield, flow-through is transferred back into column and spun down at different rpm.
Discard flow-through, add 400ul W1 buffer, and centrifuge at full speed for 30s.
Discard flow-through, add 600ul Wash Buffer buffer, and centrifuge at full speed for 30s.
Discard flow-through and centrifuge to dry column at full speed for 3min.
Replace column in 1.5ml centrifuge tube.
Add 40ul Elution Buffer and let it stand for 2min.
Centrifuge at full speed for 1min.
Perform Nanodrop to determine DNA concentration.
Double Digestion
Material
Fermentas FastDigest Restriction Enzymes and Buffers
PCR Strip Tubes
Procedures
Add 2ul 10X FastDigest Green buffer to PCR tube.
Add an amount containing 1ug of DNA sample. If DNA concentration is <62.5ng/ul, add 16ul.
Dissolve agarose by heating the solution with a microwave oven. Pour the fully dissolved agarose solution in mold, insert comb and let solidify at room temperature.
After agarose gel is ready, remove comb and transfer the gel into electrophoresis machine.
Add TAE to immerse the gel.
Load sample into gel and run electrophoresis for 40min at a voltage 10X length of gel in cm.
Gel extraction
Material
ThermoFisher Gel Extraction Kit
Procedures
Excise the desired band from gel with 4 straight cuts at each side of the band. Place the band in a 1.5ml tube.
Add 1:1 weight(mg):volumn(ul) Binding Buffer.
Incubate at 55°C for about 10min, until gel is fully dissolved.
Transfer dissolved solution to filter column, centrifuge at 6000rpm for 1min.
Repeat last step a few more times, with gradually increasing rotating speed until full speed.
Discard flow-through, add 700ul Wash Buffer and centrifuge at full speed for 1min.
Discard flow-through and centrifuge to dry column at full speed for 1min.
Replace column in 1.5ml centrifuge tube.
Add 30ul Elution Buffer and let it stand for 2min.
Centrifuge at full speed for 1min.
Store DNA at -20°C if not using it immediately.
ligation
Material
Fermentas Ligation Kit
PCR Strip Tubes
MQ water
Procedures
Measure concentraion of insert and vector
Add at least 50ng of vector
Add insert in 1:5 vector to insert ratio
Add 2uL of 10X T4 Ligase Buffer
Add 1uL of T4 Ligase
Top up till 20uL with MQ water
Incubate at room temperature for 2 hours
transformation
Material
Competent Cells
Heating Block
LB Medium
LB Antibiotic Agar Plate
Spreader
Procedures
Thaw Competent Cells on ice.
Add 10ul ligated product into Competent Cell and quickly place cell back on ice, incubating for 20mins.
Conduct heat shock at 42°C for 90s.
Chill cell on ice for 5min.
Add 900ul LB to cell.
Incubate at 30/37°C for 1h.
Spread 100ul of culture on LB antibiotic agar plate.
Culture overnight at 37°C.
Store plate at 4°C.
conjugation
Material
MW3064 Overnight Culture
MR-1 Overnight Culture
LB Medium
LB Antibiotic Agar Plate
Procedures
Take 100ul of MW3064 and MR-1 culture each and mix in centrifuge tube.
Centrifuge at 6000rpm for 3 minutes.
Discard the supernatant.
Resuspend the cell pellet with 1ml LB medium.
Let it stand for 30 minutes at 30°C.
Streak on LB antibiotic agar plate.
Culture overnight at 30°C.
growth curve measurement
Material
M1 media
MQ water
Bio-Rad Microplate Reader
96-well microplate
50ml Falcon Tube
Procedures
Inoculate strains for overnight culture in 5mL LB at 30C
Measure the OD600 of the overnight culture with 200uL of sample
Dilute OD600 to 0.001 in 50mL of M1 media and incubate in 30C
Take OD600 measurements at 2 hours intervals by taking 200uL samples from the culture media
Take samples at 2 hour intervals for 24 hours after that sample at 4 hour intervals for 12 hours
Fluorescence measurement
Material
LB
MQ water
Micro-centrifuge
96-well microplate
50ml Falcon Tube
Tecan Infinite m200 microplate reader
PBS solution
Procedures
Inoculate strains for overnight culture in 5mL LB
Measure the OD600 of the overnight culture with 200uL of sample
Dilute OD600 to 0.05 in 5mL of LB in 15mL tube
Incubate at 30C for 4 hours so that bacteria reaches mid-log phase
Dilute OD600 to 0.05 in 10mL of LB in 50mL tube
Take 400uL of samples from culture media at one hour intervals starting from T=1hr
Centrifuge at 15000rpm for 3 minutes
Discard supernatant and resuspend pellet with 400uL PBS