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. It generates the long linear concatamers of the DNA from circular templates.
  • 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. bellow is our protocol
  • 1. Digest insert and vector with Fermentas Fast enzymes
  • insert with XBAI and at the same time add CIAP(Digest 1: 3ul reaction buffer; 1restriction enzyme; 5 ul DNA; 20ul water and 1ul CIAP), deactivate enzymes at 65C for 20 min, then digest insert with PSTI(simply add 1ul of the enzyme).
  • vector with PSTI and at the same time add CIAP(Digest 1: 3ul reaction buffer; 1restriction enzyme; 5 ul DNA; 20ul water and 1ul CIAP), deactivate enzymes at 80C 20 min, then digest insert with SPEI(simply add 1ul of the enzyme)
  • Prepare Lithium-botare agarose gel (1L 20X LB gel buffer=>950 mL of dH2O, 8.392 g lithium hydroxide monohydrate; 36 g of boric acid; pH 8.2. Volume to 1 L). Prepare gel as with TE or TBE, but you can run it on much higher voltage we use 300-400V and gel is ready in 15 minutes. Theoretically maximum you can use is max. 100V/cm, but we never tried that much.
  • Run the DNA on the gel and extract insert and vector
  • Ligate:7 ul H20; 5 ul insetr; 5 ul vector; 2 ul buffer; 0.5 ul Ligase incubate for 15 min at 25C
  • Perform exonuclease digestion (2,5 recBCD exonuclease buffer; 2ul ATP; 1ul recBCD (Epicentre) ; 1 exonuclease I (Fermentas); 1 ul ligation mixture; 17,5 h20. Inactivate the reaction at 80C for 20 min

Results of the cell-free cloning
Angry face

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