Design Primers

1. Create the gene sequence using KEGG and ApE
2. Design primers using IDT's Oligo Analyzer
Considerations:
• Primers should be at least 40 base pairs (bp) long and contain approximately 20 bp from each joining fragment
• For each primer pair (that is, primers amplifying the same gene), the melting temperature (Tm) of the entire primer should be close as well as the 2nd half Tm
• The highest hairpin Tm should be less than 50 °C
• Avoid repeats of 4 or more
• At the 3' end, you should end with a G or C, avoid a T or mismatches, and avoid runs of 3 or more G/Cs in the last 5 bps at the 3' end
• G/C content should be 40-60%

PCR Gibson Fragments

1. For genes in plasmids:
• Start overnight cultures from glycerol stocks
• Miniprep overnight cultures
• PCR using mini prepped plasmid DNA
• Add all components as described in Table 1-1



Table 1-1: PCR Mixture
Component	Volume (μL)
5X Phusion Buffer	10
10mM dNTPs	1
Primer A	2.5
Primer B	2.5
Template DNA	01
Phusion DNA Polymerase	0.5
Nuclease-Free Water	Fill to 50 μL
Total	50



• Pipette up and down to mix
• Run PCR Thermalcycler Program as described in Table 1-2

Table 1-2: PCR Thermalcycler Program
Step	Temperature (C)	Time	Cycles
Initial denaturation	98	30 s	1
Denaturation	98	10 s	5
Annealing	Lower 2nd half Tm + 3	15 s	5
Extension	72	30 s/1 kb	5
Denaturation	98	10 s	25
Anneal + Extension	72	30 s/1 kb	25
Final Extension	72	10 min	1
Final Hold	4	hold	2-



• Save 5 μL PCR product for gel electrophoresis
• Digest with DpnI to remove methylated DNA as described in Manual
2. For genes not in plasmids:
• Follow genomic DNA PCR thermal cycler protocol (see Tables 1-1 and 1-2)

Check and Clean Up PCR Products

1. Check size of each PCR product by gel electrophoresis
2. If any PCR reaction was unsuccessful, repeat the PCR of the Gibson Fragments
3. Clean up PCR products of correct size with the PCR clean-up kit and determine DNA concentrations

Prepare Gibson Reaction

1. Use Equation in manual to calculate the fragment concentration
• For 2 - 3 fragments, the total DNA should be 0.05 - 0.2 pmols
• For 4 - 6 fragments, the total DNA should be 0.2 - 1.0 pmols
2. Make Gibson reaction mixture according to manual
• Pipette up and down to mix
3. Run thermalcycler program as described in manual

Transformation

1. 30 minutes before the end of the Gibson reaction, thaw competent cells on ice and set water bath to 42 DEGC
2. Add 2.5 uL Gibson product to cells
3. Heat shock cells for 30 seconds at 42 C without shaking
4. Place on ice for 2 minutes
5. Aseptically (in hood) add 250 uL appropriate media to the tube and cap tightly
6. Place tubes horizontally in incubator; incubate at 37 C and 225 rpm for 1 hour
7. In the hood, spread 100 uL on plate
8. Incubate overnight at 37 C; store remaining culture at 4 C

Confirm Correct Construction of Plasmid (Colony PCR)

1. Choose primers that flank multiple fragments of the assembled DNA
2. Set up PCR reaction (follow reaction mixture from above for 50 uL)
3. To add template DNA:
• Using a sterile toothpick or pipette tip, touch colony, rotate 180 degrees, and touch colony again
• Streak on LB plate with appropriate antibiotics so that you can use colony for future steps
• Swirl toothpick/pipette tip in the PCR tube to resuspend the cells
• Pipette up and down to mix
4. Run PCR thermalcycler program as described in manual.
5. Run gel electrophoresis to verify PCR product and confirm plasmid by sequencing if desired