Set up the following reaction On ice:

A	B	C
COMPONENT	25 µl REACTION	FINAL CONCENTRATION
5X Q5 Reaction Buffer	5 µl	1X
10 mM dNTPs	0.5 µl	200 µM
10 µM Forward Primer	1.25 µl	0.5 µM
10 µM Reverse Primer	1.25 µl	0.5 µM
Template DNA	variable	~ 5 ng
Q5 High-Fidelity DNA Polymerase	0.25 µl	0.02 U/µl
5X Q5 High GC Enhancer (optional)	(5 µl)	(1X)
Nuclease-Free Water	to 25 µl

Gently mix the reaction.

Collect all liquid to the bottom of the tube by a quick spin if necessary.

Quickly transfer PCR tubes to a PCR machine preheated to the denaturation temperature (98°C) and begin thermocycling.

Thermocycling Conditions for a Routine PCR:

A	B	C
STEP	TEMP	TIME
Initial Denaturation	98°C	30 seconds
25–35 Cycles	98°C	5–10 seconds
	*50–72°C	10–30 seconds
	72°C	20–30 seconds/kb
Final Extension	72°C	2 minutes
Hold	4–10°C

*Use of the NEB Tm Calculator is highly recommended.

Digest the lead gene templates with DpnI at 37°C for 2 hours. Heat-inactivate at 80°C for 20 minutes.

Perform rapid PCR cleanup (see: http://dx.doi.org/10.17504/protocols.io.bg9xjz7n)

Perform PCR DNA cleanup (see: http://dx.doi.org/10.17504/protocols.io.bg9rjz56)

Quantify DNA concentration by Qubit Fluorometer (Qubit 2.0 Fluorometer, ThermoFisher Scientific).

Measure DNA length using TapeStation (Agilent 2200 TapeStation).

Follow the steps for Amplify constant fragments (steps 1-9), skipping step 5.

Combine variable fragments of the same segment in equal concentration.

Set up 25 µl assembly reactions as follows:

A	B
REAGENTS	ASSEMBLY REACTION
DNA inserts 100 ng/ul each	1 µl (~ 100 ng) each, (up to 39 µl)
T4 DNA Ligase Buffer (NEB #B0202) (10X)	5 μl
T4 DNA Ligase (NEB #M0202), 2000 U/µl	1 μl (2000 units)
BsaI-HFv2 (NEB #R3733), 20 U/µl	3 μl (60 units)
Nuclease-free H2O (NEB #B1500)	to 50 µl

Mix gently by pipetting up and down 4 times.

Briefly microcentrifuge (1 second) to bring material to the bottom of tube.

Transfer to thermocycler and program as follows: (5 min 37ºC → 10 min 16ºC) x 60 cycles followed by 5 minutes 60ºC. If reactions are done overnight, add a 4ºC terminal hold to the protocol, but repeat the final 5 minutes 60ºC step the next day before the transformations.

Perform PCR DNA Cleanup (http://dx.doi.org/10.17504/protocols.io.bg9sjz6e) or follow a size selection protocol.

For 1x reaction set up the following reaction On ice:

A	B	C
Component	25 µl Reaction	Final Concentration
Q5 High-Fidelity 2X Master Mix	12.5 µl	1X
10 µM Forward Primer	1.25 µl	0.5 µM
10 µM Reverse Primer	1.25 µl	0.5 µM
GG assembly products	5 µl
Nuclease-Free Water	to 25 µl

Gently mix the reaction.

Collect all liquid to the bottom of the tube by a quick spin if necessary.

Quickly transfer PCR tubes to a PCR machine preheated to the denaturation temperature (98°C) and begin thermocycling.

Thermocycling Conditions for a Routine PCR:

A	B	C
STEP	TEMP	TIME
Initial Denaturation	98°C	30 seconds
25–35 Cycles	98°C	5–10 seconds
	*50–72°C	10–30 seconds
	72°C	10 seconds/kb
Final Extension	72°C	2 minutes
Hold	4–10°C

*Use of the NEB Tm Calculator is highly recommended.

Perform Oligonucleotide Cleanup (http://dx.doi.org/10.17504/protocols.io.bg9sjz6e) or follow a size selection protocol.

Quantify DNA concentration by Qubit.

Measure DNA length using TapeStation.