Team:Warsaw/SyntheticCloning/SyntheticCloning

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Example Tabs

Step by step guide to synthetic cloning


Rationale behind the protocol
  • 1. Cut DNA with one of the enzyme you want to use in cloning
  • 2. Dephosphorylate 5' phosphate - this prevents DNA pieces from self-ligation
  • 3. Cut DNA with the other of the enzyme you want to use in cloning
  • 4. Run DNA on the gel
  • 5. Extract vector and insert from the gel.
  • 6. Ligate
  • 7. After ligation some linear DNA may still remain, you can get rid of it by using recBCD exonuclease and Exonuclease I
  • Linear DNA is a perfect template for RCR amplification with phi 29 polymerase.
  • After 12 hours of amplification DNA can be used in downstream processing
An excelent protocol on how to perform cell-free cloning is available here: Takahashi H, Yamamoto K, Ohtani T, Sugiyama S. Cell-free cloning using multiply-primed rolling circle amplification with modified RNA primers. Biotechniques. 2009 Jul.

Results of the cell-free cloning
Angry face

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