Team:TU Delft/protocols/ligation

From 2010.igem.org

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(New page: ==Ligation== Ligations (pasting plasmid DNA) were performed at the appropriate temperature with the appropriate buffer in the appropriate concentration, according to the supplier. React...)
 
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==Ligation==
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=Ligation=
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Ligations (pasting plasmid DNA) were performed at the appropriate temperature with the appropriate buffer in the appropriate concentration, according to the supplier.
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''Materials:''
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- digested plasmid DNA or PCR product
 +
 +
- T4 ligation buffer (10x) (Fermentas or BioLabs)
 +
 +
- T4 ligase (Fermentas or BioLabs)
 +
 +
- H2O
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 +
- water bath at 16 °C
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 +
 +
''Protocol:''
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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:
Reaction for one sample:
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|-
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|DNA vector
|DNA vector
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|x μL (for 1×)
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|x μL  
|-
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|T4 Ligation buffer (10×)
|T4 Ligation buffer (10×)
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|2.5 μL (for 1×)
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|x μL (for 1×)
|-
|-
|T4 Ligase
|T4 Ligase
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|x μL
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|1.0 μL
|-
|-
|H2O
|H2O
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|-
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|25,0 μL (final contration ~100 ng/μL)
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|10-15 μL
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The final concentration is preferably ~100 ng/μL. Incubate at 16 °C for at least 3 hours.
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For transformation use circa half of the ligation mix.
Transform circa half of the ligation mix. Incubate at 16 °C o/n
Transform circa half of the ligation mix. Incubate at 16 °C o/n

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

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