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Team:Warsaw/SyntheticCloning/SyntheticCloning
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Revision as of 22:01, 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. It generates the long linear concatamers of the DNA from circular templates.
- After 12 hours of amplification you have the DNA of your construct
This is how we did it
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. Gels in LB buffer can be can run 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.
- (1L 20 times concentrated 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)
- 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
- 3. Set up the annealing mix as described:
- 1ul of the reaction mix after exonuclease treatment
- 1ul Phi29 polymerase buffer
- 1ul PTO(phosphothioate protected) random RNA hexamers - 400umol, you can get them from IBA-GO
- RNAse free water to 10ul
- 4. Set up the annealing reaction:
- heat mix to 95C
- cool down to 95C, slowly 0.1C/s works
- 5. to the annealing mix add :
- 1ul 10umol DNTPs - it is best to use RNAse free
- 1ul phi29 polymerase from Epicentre
- 2ul phi 29 buffer
- 1ul diluted Pyrophosphatase from Fermentas. Diluted 1:100 according to the manual
- RNAse free water to 30ul final volume
- 6. Incubate at 30C for at least 2h
Results of the cell-free cloning
