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| | [[UCL_London/Protocol/Competent Bacteria |Competent Bacteria]] | | [[UCL_London/Protocol/Competent Bacteria |Competent Bacteria]] |
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| - | :'''''Materials:'''''
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| - | * 5×M9 salts in 500ml dH2O:
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| - | ** Na2HPO4--- 32g
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| - | ** KH2PO4 --- 7.5g
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| - | ** NaCl --- 1.25g
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| - | ** NH4Cl --- 2.5g
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| - | * Minimal media: ( In 50ml Falcon )
| + | [[UCL_London/Protocol/Transformation | Transformation]] |
| - | ** Melted bacteriological agar solution (< 50°C) --- 39ml
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| - | ** 5×M9 salt solution --- 10ml
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| - | ** 20% (w/v) D-glucose --- 1ml
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| - | ** 1M CaCl2 --- 5µl
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| - | ** 1M MgSO4 --- 100µl
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| - | * 0.1M CaCl2 / 15% glycerol: (In 59 ml Falcon)
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| - | ** 1M CaCl2 --- 5ml
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| - | ** 100% glycerol --- 7.5ml
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| | | | |
| - | : '''''Preparation:'''''
| + | [[UCL_London/Protocol/Glycerol Stock|Glycerol Stock]] |
| - | :: Pour minimal media plates.
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| - | :: 5x M9 salts.
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| - | :: Prepare 100ml LB per strain.
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| - | :: Prepare 50ml ice cold 0.1M CaCl2 / 15% glycerol per strain
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| - | :: Pre-chill eppendorf tubes
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| - | : '''''Method:'''''
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| - | # Streak cells on minimal agar plate. Incubate 37°C overnight.
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| - | # Pick a colony into 5 ml LB + 100µl 1M MgSO4. Incubate 37°C overnight.
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| - | # Inoculate 100ml LB in pre-warmed conical with 1ml of the 5ml O/N culture from Step 2.
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| - | # Incubate 2 hrs in 37°C shaker until the cells at early log phase of growth curve (A600 ~ 0.3).
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| - | # Transfer to chilled, sterile two 50ml Falcon tubes and incubate on ice for 10 min.
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| - | # Cf 3300g 5 min in bechtop RmT. Cf.
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| - | # Resus in 10ml ice cold 0.1M CaCl2 / 15% glycerol and incubate on ice 30 min.
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| - | # Cf 3300g 5 min in benchtop RmT. Cf.
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| - | # 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/Miniprep|Miniprep]] |
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| - | ----
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| - |
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| - | [[UCL_London/Protocol/Transformation | Transformation]]
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| | | | |
| - | : '''''Materials:'''''
| + | [[UCL_London/Protocol/Restriction Enzyme Digestion|Restriction Enzyme Digestion]] |
| - | * Plasmids DNA
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| - | * Competent E.coli
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| - | * NZY Medium
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| - | * LB buffer
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| - | * Ampicllin, Kanamycin, Tetracycline
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| - | * Agar Plates
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| - | * Eppendorf
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| - | : '''''Method 1:'''''
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| - | # Add 5µL ice-cold plasmid DNA into the competent E.coli on ice.
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| - | # Incubate on ice for 30 min. (minimum) Incubate at 42°C for 45 sec.
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| - | # Incubate on ice for 2 min. (minimum)
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| - | # Add 300µL of NZYX medium.
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| - | # Incubate with shaking at 37°C for 1 hr.
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| - | # Spin the incubated E.coli suspension for 3min at maximum speed. (Optional)
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| - | # Discard about half of the liquid, and then re-suspend the E.coli solution. (Optional)
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| - | # Spread the suspension onto selective agar plates.
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| - | # Incubate at 37°C overnight.
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| - | # Pick a colony into 2ml selective LB (Add 100µL antibiotic to 50mL LB medium). Incubate 37°C in shaker overnight
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| | | | |
| - | : '''''Method 2:'''''
| + | [[UCL_London/Protocol/Gel Electrophoresis|Gel Electrophoresis]] |
| - | # Using the puch tool, puch out the appropriate DNA. Clean the tool between punches.
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| - | # 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.
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| - | # Add 2µL of DNA in TE to 50µL of competent cells (TOP10)
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| - | # Allow the DNA and competent cells to sit on ice for 30 minutes
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| - | # Heat shock at 42ºC for 60 sec in water bath.
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| - | # Recover on ice for 5 min.
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| - | # Add 200 µL SOC media.
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| - | # Incubate at 37ºC for 2 hr while the tubes are rotating.
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| - | # Centrifuge and leave about 100 µL liquid; hence, resuspend the E.coli suspension.
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| - | # Plate 250µL on an LB plate with the appropriate antibiotic.
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