Team:TU Darmstadt/Protocols/

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Ligation reaction mix

10x NEB 4 Buffer 2µL

T4 Ligase 1µL

Vector 50 ng

Insert X

H2O ad 20µL

The insert to vector molar ration of 1:5 was chosen and caltulated with the formula:

A ligation with T4 DNA Ligase(purchased from NEB) followed.

Schmidt´sche thermocycler ligation program (STLP)

1. T= 4.0°C 10 min
2. T= 10°C 60 min
3. T= 12°C 15 min
4. T= 16°C 10 min
5. T= 25°C 10 min
6. T= 37°C 5 min
7. T= 70°C 20 min

References

• Lund, A. H., M. Duch, et al. (1996). "Increased cloning efficiency by temperature-cycle ligation." Nucleic Acids Res 24(4): 800-801.
• Der Experimentator: Molekularbiologie / Genomics/6.Auflage/2009/C.Mülhardt