Team:UCL London/From the lab/Protocols

[[UCL_London/Protocol/Competent Bacteria |Competent Bacteria]]

:'''''Materials:'''''
* 5×M9 salts in 500ml dH2O: 
** Na2HPO4--- 32g
** KH2PO4 --- 7.5g
** NaCl --- 1.25g
** NH4Cl --- 2.5g

* Minimal media: ( In 50ml Falcon ) 
** Melted bacteriological agar solution (< 50°C) --- 39ml
** 5×M9 salt solution --- 10ml
** 20% (w/v) D-glucose --- 1ml
** 1M CaCl2 --- 5µl
** 1M MgSO4 --- 100µl

* 0.1M CaCl2 / 15% glycerol: (In 59 ml Falcon)
** 1M CaCl2 --- 5ml
** 100% glycerol --- 7.5ml

: '''''Preparation:'''''
:: Pour minimal media plates.
:: 5x M9 salts.
:: Prepare 100ml LB per strain.
:: Prepare 50ml ice cold 0.1M CaCl2 / 15% glycerol per strain
:: Pre-chill eppendorf tubes

: '''''Method:'''''
# Streak cells on minimal agar plate. Incubate 37°C overnight.
# Pick a colony into 5 ml LB + 100µl 1M MgSO4. Incubate 37°C overnight.
# Inoculate 100ml LB in pre-warmed conical with 1ml of the 5ml O/N culture from Step 2.
# Incubate 2 hrs in 37°C shaker until the cells at early log phase of growth curve (A600 ~ 0.3).
# Transfer to chilled, sterile two 50ml Falcon tubes and incubate on ice for 10 min.
# Cf 3300g 5 min in bechtop RmT. Cf.
# Resus in 10ml ice cold 0.1M CaCl2 / 15% glycerol and incubate on ice 30 min.
# Cf 3300g 5 min in benchtop RmT. Cf.
# Resus in 1ml ice cold 0.1M CaCl2 / 15% glycerol. Transfer 100µl aliquots to pre-chilled, pre-labelled eppendorf tubes. Store -80°C.


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[[UCL_London/Protocol/Transformation | Transformation]]

: '''''Materials:'''''  
* Plasmids DNA
* Competent E.coli
* NZY Medium
* LB buffer
* Ampicllin, Kanamycin, Tetracycline
* Agar Plates
* Eppendorf 

: '''''Method 1:'''''
# Add 5µL ice-cold plasmid DNA into the competent E.coli on ice.
# Incubate on ice for 30 min. (minimum) Incubate at 42°C for 45 sec.
# Incubate on ice for 2 min. (minimum)
# Add 300µL of NZYX medium.
# Incubate with shaking at 37°C for 1 hr.
# Spin the incubated E.coli suspension for 3min at maximum speed. (Optional)
# Discard about half of the liquid, and then re-suspend the E.coli solution. (Optional)
# Spread the suspension onto selective agar plates.
# Incubate at 37°C overnight.
# Pick a colony into 2ml selective LB (Add 100µL antibiotic to 50mL LB medium). Incubate 37°C in shaker overnight

: '''''Method 2:'''''
# Using the puch tool, puch out the appropriate DNA. Clean the tool between punches. 
# Soak the spots in 5µL of TE that has been warmed to 50ºC for 20 minutes. At this time take the competent cells from the -80º freezer and start defrosting on ice. 
# Add 2µL of DNA in TE to 50µL of competent cells (TOP10) 
# Allow the DNA and competent cells to sit on ice for 30 minutes 
# Heat shock at 42ºC for 60 sec in water bath. 
# Recover on ice for 5 min. 
# Add 200 µL SOC media. 
# Incubate at 37ºC for 2 hr while the tubes are rotating. 
# Centrifuge and leave about 100 µL liquid; hence, resuspend the E.coli suspension.
# Plate 250µL on an LB plate with the appropriate antibiotic.