16. Incubate the tube at 37
C for 60 min with vigorously shaking
(250 rpm).
17. Spread 100 μL of the cells and DNA mixture onto an LB/ampicillin plate.
18. Incubate overnight at 37
C.
19. Day 2: Perform colony PCR to check the size of the insert
DNA. Prepare the colony PCR mix (10 μL) in 0.2 mL PCR
tubes as in Table 8.
20. To create a stock of each individual colony, pick a small colony
using a sterile pipette tip, streak the pipette tip onto another
LB/ampicillin plate, and grow at 37
C for more than 6 h.
Table 7
Gibson assembly reaction
Component
Size of DNA fragment
Amount per reaction
Fab L
700 bp
0.5 pmol
a
iRATmC
980 bp
0.5 pmol
a
Fab H
700 bp
0.5 pmol
a
VEC-FB
4800 bp
0.16 pmol
a
Gibson Assembly Master Mix (2Â)
–
1 0μL
Sterile MilliQ water
–
To make a final volume of 20 μL
Total
20 μL
a
pmol ¼ (weight in ng)  1000/(base pairs  650 Da)
Table 8
Colony PCR mix
Component
Volume/reaction
Final concentration
H 2 O
3.9 μL
– –
10Â Ex Taq Buffer
1 μL
1 Â
dNTPs mixture
(2.5 mM each)
1 μL
250 μM each
SFseq-F (1 pmol/μL)
2 μL
0.2 μM
SFseq-R (1 pmol/μL)
2 μL
0.2 μM
TaKaRa Ex Taq (5 U/μL)
0.1 μL
0.05 U/μL
Total
10 μL
94
Norimichi Nomura et al.