Team:TU Delft/protocols/ligation

From 2010.igem.org

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==Ligation==
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=Ligation=
''Materials:''
''Materials:''
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- digested plasmid DNA or PCR product
- digested plasmid DNA or PCR product
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- T4 ligation buffer (10x) (Fermentas)
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- T4 ligation buffer (10x) (Fermentas or BioLabs)
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- T4 ligase (Fermentas)
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- T4 ligase (Fermentas or BioLabs)
- H2O
- H2O
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|T4 Ligation buffer (10×)
|T4 Ligation buffer (10×)
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|2.0 μL (for 1×)
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|x μL (for 1×)
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|T4 Ligase
|T4 Ligase

Latest revision as of 18:52, 12 September 2010

Ligation

Materials:

- digested plasmid DNA or PCR product

- T4 ligation buffer (10x) (Fermentas or BioLabs)

- T4 ligase (Fermentas or BioLabs)

- H2O

- water bath at 16 °C


Protocol:

Ligations (pasting plasmid DNA) were performed at the appropriate temperature with the appropriate buffer in the appropriate concentration, according to the supplier.

Reaction for one sample:

DNA insert x μL
DNA vector x μL
T4 Ligation buffer (10×) x μL (for 1×)
T4 Ligase 1.0 μL
H2O x μL
10-15 μL

The final concentration is preferably ~100 ng/μL. Incubate at 16 °C for at least 3 hours. For transformation use circa half of the ligation mix.

Transform circa half of the ligation mix. Incubate at 16 °C o/n