A PCR reaction to generate linearized construction vector backbones for Fusion(Freiburg)-formatted Biobrick assemblies.
The classic assembly protocol digests the target vector (usually pSB1*) in parallel to the digestions of the two Biobrick parts. We prefer to create a stock of target backbone by PCR with a fast high-fidelity polymerase. This way, mutations in the ccdB death cassette of the target vector cannot escape selection.
- 100 ul + PCR tubes
- Phusion HotStart Polymerase 2 U/ul
- Phusion HF Buffer 5x
- dNTP mix 10mM each nucleotide
- reverse prefix primer rg0301 (CRG) -- -- BBa_J18910 (RFC 25 format, ACCGGTTAATACTAGTAGCGGCC)
- reverse suffix primer rg0302 (CRG) -- BBa_J18911(RFC 25 format, GCCGGCCATCTAGAAGCG)
- vector template DNA
- DpnI restriction enzyme
- Antarctic Phosphatase and 10 x buffer
setup PCR reaction
|µl, single reaction||3.5xMaster||10xMaster|
|5x HF Buffer||20µl||70||200|
|rg0301 100 µM||0.5µl||1.75||5|
|rg0302 100 µM||0.5µl||1.75||5|
- 35x (10"@98°C; 15"@68C; 1'30"@72C);
- inf. 4C
- add 1µl DpnI, incubate for 1h @ 37°C
- mix the tube so that the enzyme contacts all surfaces
- longer (2h) incubation may still reduce background
- heat-inactivate 20'@80°C
- purify with PCR purification kit
- elute in water **not** elution buffer
- dilute to 28 ng/µl (we assume a 10 % dilution by the following Phosphatase treatment)
- proceed with restriction C
- add 2µl restriction mix C to each 8µl DNA
- incubate for 3h @ 37°C
- heat-inactivate 20' @ 80°C
- proceed with phosphatase treatment
- add 10 x antarctic phospatase buffer to 1 x final concentration
- add 1µl Antarctic Phosphatase
- incubate 1h @ 37°C; heat-inactivate 5' @ 65°C
- verify samples on 1% Agarose gel
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
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