3A Assembly

1. Digestion

A 25 µL master mix was prepared for both of the inserts. The backbone plasmid was already precut, but to cut backbone plasmids EcoRI-HF and PstI would be used (the same volume).

Enzyme Master Mix for the first insert:

NEB Buffer 2	5 µL
EcoRI-HF	0.5 µL
SpeI	0.5 µL
Milliq	19 µL

4 µL of the master mix was then mixed with 4 µL of the enzyme.

Enzyme Master Mix for the second insert:

NEB Buffer 2	5 µL
XBaI	0.5 µL
PstI	0.5 µL
Milliq	19 µL

4 µL of the master mix was then mixed with 4 µL of the enzyme.

Both digestion mixtures were digested at 37 °C for 30 minutes. The mixtures were then left at 80 °C for 20 minutes to heat kill the enzymes. These steps were performed in a PCR machine.

2. Ligation

In this step it is important to have equimolar amount of cut plasmid backbone and digest product. In our case the length of everything was very similar, therefore the same volumes could be used, however that can always be done. The total reaction volume is 10 µL

Ligation Mixture:

Plasmid backbone	2 µL
Enzyme insert cut at E and S	2 µL
Enzyme insert cut at X and P	2 µL
T4 DNA Ligase Buffer	1 µL
T4 DNA Ligase	0.5 µL
MilliQ	2.5 µL

The mixture was then ligated at 16 °C for 30 minutes. Another heat kill around at 80 °C for 20 minutes was then performed.

For the transformation step see the Phusion PCR, Gibson Assembly and Transformation protocol.

Phusion PCR, Gibson assembly and Transformation

1. Phusion PCR

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

Phusion PCR reaction mixture:

Plasmid	…. µL (~30 pg)
5X Phusion HF Buffer	10 µL
dNTP (10 mM)	<1 µL/td>
Reverse Primer (10 µM)	2.5 µL
Forward Primer (10 µM)	2.5 µL
Phusion DNA pol	0.5 µL
DMSO	1.5 µL
H₂O	up to 50 µL
Total volume	50 µL

PCR reaction conditions:

1) Initial denaturation:	98 °C for 30 seconds
2) Denaturation in following cycles:	98 °C for 10 seconds
3) Primer annealing:	Temperature gradient between 58–65 °C for 30 seconds
4) Extension:	72 °C 1.5 minutes
5) Final DNA synthesis:	72 °C for 5 minutes

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 :

Plasmid PCR fragment	.… µL (~0.02 pmol)
Gibson fragment	.… µL (~0.06 pmol)
Gibson Assembly Master Mix (2x)	10 µL
H₂O	up to 20 µL
Total volume	20 µL

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.

Team:Uppsala/Experiments