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Team:Warsaw/SyntheticCloning/SyntheticCloning
From 2011.igem.org
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<li>After 12 hours of amplification DNA can be used in downstream processing</li> | <li>After 12 hours of amplification DNA can be used in downstream processing</li> | ||
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| - | + | An excelent protocol on how to perform cell-free cloning is available here: <a href="http://www.biotechniques.com/multimedia/archive/00051/BTN_A_000113155_O_51382a.pdf" target="_blank">Takahashi H, Yamamoto K, Ohtani T, Sugiyama S. <i>Cell-free cloning using multiply-primed rolling circle amplification with modified RNA primers.</i> Biotechniques. 2009 Jul. | |
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Revision as of 20:39, 20 September 2011
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
Results of the cell-free cloning
