Team:Tuebingen/Notebook/Protocols/3a-assembly

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3A-Assembly

Digestion

Reagents

2.5 µL 10x NEBuffer 2
0.25 100x BSA
250 - 300 ng Plasmid DNA
0.5 µL EcoRI or XbaI
0.5 µL PstI or SpeI
to 25 µL Aqua dest.
1/10 vol 10X Antarctic Phosphatase

 

Procedure

  1. Mix all reagents (except for Antarctic Phosphatase) in an 1.5 mL Eppendorf-tube and incubate at 37 °C over night.
  2. On the next day, heat inactivate restriction enzymes at 80°C for 20 min.
  3. Add 1/10 volume of 10X Antarctic Phosphatase and mix. Incubate at 37 °C for 15 min for 5' extensions.
  4. Heat inactivate phosphatase at 70 °C for 5 min.
  5. Perform the previous steps for upstream part plasmid, downstream part plasmid, and destination part plasmid. Digest upstream part with EcoRI / SpeI, downstream part with XbaI / PstI, and destination part plasmid with EcoRI / PstI.

 

Ligation

Reagents

2.0 µL 10x T4 DNA Ligase Buffer
2.0 Upstream Part digestion
2.0 Downstream Part digestion
2.0 Destination Part digestion
2.0 µL T4 DNA Ligase
11 µL Aqua dest.

 

Procedure

  1. Mix all reagents in an 1.5 mL Eppendorf-tube and incubate at 37 °C over night.
  2. On the next day, heat inactivate restriction enzymes at 80°C for 20 min.
  3. Transform 3A-Assembly product in cells.

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