1. Phusion PCR

Total reaction volume 50 µL
Amount of plasmid per reaction ~30 pg

Phusion PCR reaction mixture:

PCR reaction conditions:

Steps 2–4: 25 cycles
Lid temperature at 105 °C

2. DpnI Treatment of the PCR Fragment

To the 50 µL PCR reaction mix from step 1 add:

2.4 µL “FastDigest” DpnI
6 µL 10X “FastDigest” Buffer

1) Incubate reaction mixture at 37 °C for 1 hour
2) Purify the DNA in the mixture with “PureLink Quick PCR Purification Kit” (Invitrogen). Milliq was used instead of TE buffer when eluting the DNA from the spin column in the final step of the DNA purification. The optional washing step was skipped
3) Measure the DNA concentration on Nanodrop in units of ng/µL. 2 µL was used for each measurement
4) Store the DNA at -20 °C until further use

3. Gibson Assembly Reaction

1. Mix PCR fragment from the plasmid and the Gibson fragment containing the insert. The total volume is 20 µL:

Mix i) the PCR fragment made from the plasmid and ii) the Gibson fragment containing the gene of interest. Total volume of the Gibson reaction mix is 20 uL :

2. Incubate the mixture at 50 °C for 1 hour

4. Transformation

1. Take out competent cells (we used Escherichia Coli TOP10 for cloning and E. coli BL21 (DE3*) for protein expression from Invitrogen) from the -80 °C and thaw them on ice from approximately 15 minutes.
2. Add 2 µL of the Gibson mixture/Ligation mixture to the competent cells or ~7.5 ng of gBlocks.
3. Incubate on ice for 30 minutes.
4. Incubate at 42 °C for 30 seconds (water bath).
5. Incubate on ice for 5 minutes.
6. Add 200 µL of LB to the mixture and incubate it at 37 °C with 200 rpm for 1 hour.
7. Plate the mixtures on chloramphenicol plates (200 µL 1:1 dilution and 200 µL 1:100 diluted with LB). Beads were used to spread the mixture on the plates (10–20 beads).
8. Incubate plates overnight at 37 °C.