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It is most likely that your DNA will not already overlap, so you'll need to add the overlap yourself. A simple PCR with a primer comprised of two bits of DNA - one from the target DNA (the annealing portion) and one from the adjacent DNA (the flappy end).
This means, to join two strands of DNA (A and B) with an overlap of 40, A will need to be extended as follows
These are all written and read 5' to 3', and the primers for B are more or less the reverse complement of the primers for A.
This is better explained with an example, using an overlap of 8
Strand A: 5' ATGGTACCGGA 3' Strand B: 5' GGTACCAGTTC 3' 3' TACCATGGCCT 5' 3' CCATGGTCAAG 5'
3' GCCTCCAT 5' 5' ATGGTACCGGA 3' 3' TACCATGGCCT 5' 5' GTTCATGG 3'
Strand A: Top: 5' TACCGCCT 3' Bottom: 5' GTTCATGG 3' Strand B: Top: 5' CGGAGGTA 3' Bottom: 5' CCATGAAC 3'
It is likely that you will be using DNA that is already in a vector of some sorts - the procedure is exactly the same as the diagrams below show:
(1) PCR cycle 1: melting
A single strand of the vector containing RFPm
(2) PCR cycle 1: annealing
One of the Gibson primers attaches by the annealing portion to the RFP. The flappy end does nothing.
(3) PCR cycle 1: extension
The primer is extended around the full vector.
(4) PCR cycle 2: melting
We now have a linear single strand of DNA with one extension.
(5) PCR cycle 2: annealing
The other Gibson primer attaches by the annealing portion to the other end of the RFP. The flappy end does nothing.
(6) PCR cycle 2: extension
The primer is extended to the end of the RFP plus the short extension for the overlap.
(7) All further PCR cycles
PCR now continues with the Gibson primers acting as normal primers. Both the annealing portions and flappy ends anneal to the extended DNA.
The principle at one join is as follows, with the two strands in black and red, and enzyme action in blue:
5'...ATCGAGGCTGTTAGGAGTATTACGTATTCGAGGATTCGAGC 3' 3'...TAGCTCCGACAATCCTCATAATGCATAAGCTCCTAAGCTCG 5' 5' TACGTATTCGAGGATTCGAGCAGTCGATCAGGATTCGATTC...3' 3' ATGCATAAGCTCCTAAGCTCGTCAGCTAGTCCTAAGCTAAG...5'
Stage 1: two DNA strands with a 40bp overlap (shown here as 20bp)
5'...ATCGAGGCTGTTAGGAGTATTACGTATTCGAGGATTCGAGC 3' 3'...TAGCTCC 5' <-- exonuclease exonuclease --> 5' TCGATTC...3' 3' ATGCATAAGCTCCTAAGCTCGTCAGCTAGTCCTAAGCTAAG...5'
Stage 2: As the sample is heated to 50°C, an exonuclease chews back 5' to 3'
polymerase 5'...ATCGAGGCTGTTAGGAGTATTACGTATTCGAGGATTCGAGC 3' --> 5' TCGATTC...3' 3'...TAGCTCC 5' <-- 3' ATGCATAAGCTCCTAAGCTCGTCAGCTAGTCCTAAGCTAAG...5' polymerase
Stage 3: At 50°C, the sticky ends anneal, and a polymerase begins repairing the gap 3' to 5'
ligase 5'...ATCGAGGCTGTTAGGAGTATTACGTATTCGAGGATTCGAGCAGTCGATCAGGATTCGATTC...3' 3'...TAGCTCCGACAATCCTCATAATGCATAAGCTCCTAAGCTCGTCAGCTAGTCCTAAGCTAAG...5' ligase
Stage 4: Finally, a ligase repairs the backbone and the seamless join is complete
Both of these protocols were found to work well by the 2010 Cambridge iGEM team.
For assembly, the following was found to be highly reliable
In time, the protocol for extension will be provided in detail by Gibthon; in the mean time here is a template from which to work.
Note that Tm for the primer here refers to the annealing portion of the primer.