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| - | '''Competent Bacteria:'''
| + | {{Template:UCL_London_Header}} |
| | + | {{Template:UCL_London_Lab}} |
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| - | :'''''Materials:'''''
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| - | * 5×M9 salts in 500ml dH2O:
| + | [[UCL_London/Protocol/Competent Bacteria |Competent Bacteria]] |
| - | ** 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 )
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| - | ** 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)
| + | [[UCL_London/Protocol/Transformation | Transformation]] |
| - | ** 1M CaCl2 --- 5ml
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| - | ** 100% glycerol --- 7.5ml
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| | | | |
| - | : '''''Preparation:'''''
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| - | :: 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:'''''
| + | [[UCL_London/Protocol/Glycerol Stock|Glycerol Stock]] |
| - | # 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.
| + | [[UCL_London/Protocol/Miniprep|Miniprep]] |
| - | # 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.
| + | [[UCL_London/Protocol/Restriction Enzyme Digestion|Restriction Enzyme Digestion]] |
| - | # 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.
| + | [[UCL_London/Protocol/Gel Electrophoresis|Gel Electrophoresis]] |
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| | + | [[UCL_London/Protocol/Ligation|Ligation]] |
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| | + | </div> |
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| | + | {{Template:UCL_London_footer}} |
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