• For "intramolecular" ligation of a PCR product the 5'-ends are phosphorylated with T4 Polynucleotidekinase (PNK)
• Set up the following reaction:

reagent	volume (in µL)
10x T4 DNA ligase buffer	2.5
T4 PNK	1
PEG 4000 50%	2.5
DNA (100 to 600 ng)	x
water	to 25

• Incubate at 37 °C, 30 min
• Heat inactivate at 65 °C, 20 min
• Add 1 µL T4 DNA ligase after reaction has cooled down to room temperature
• Incubate at room temperature for at least 2 h, overnight also works.
• Next step: Transformation via heat shock

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• Thaw 50 µl electrocompetent E. coli cells on ice, dilute with icecold 50 µl glycerine (10%) if necessary
• Add 0.5-5 µl plasmid to 50 µl electrocompetent cells
• Store cells on ice for 1 minute
• Electroporate at U = 2.5 kV, C = 25 µF, R = 400 Ω
• Transfer transformation reaction to 450 µl SOC-Medium and incubate 1 h at 37 °C
• Plate on selective LB-Medium
• Incubate over night at 37 °C

• Thaw 100 µl chemo competent E. coli cells on ice
• Add 0.5-5 µl plasmid to 100 µl chemocompetent cells
• Store cells on ice for 10-30 min on ice
• Heat shock for 90 seconds at 42 °C
• Store reaction on ice for 60 seconds
• Optional: Preheat SOC medium to 37 °C
• Transfer reaction to 1 ml SOC medium and incubate at 37 °C for at least 1 hour
• Centrifuge 3 minutes at 12000 rpm and plate on selective LB medium
• Incubate at 37 °C over night

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