5. Measure the DNA concentration using a NanoDrop
spectrophotometer.
6. Set up the Gibson assembly reaction (20 μL) as in Table 7.
7. Incubate for 60 min at 50
C.
8. Add 60 μL of sterile MilliQ water to the reaction product.
9. Aliquot 10 μL of the diluted reaction product, and chill in a
1.5-mL centrifuge tube.
10. Thaw the E. coli TOP10 competent cells (100 μL) on ice.
11. Add 100 μL of competent cells to the DNA. Mix gently by
pipetting up and down to mix the cells and DNA. Do not
vortex.
12. Place the mixture on ice for 30 min.
13. Heat shock at 42
C for 30 s.
14. Place the mixture on ice for 2 min.
15. Add 900 μL of pre-warmed SOC medium to the tube.
Table 5
PCR amplification
Component
Volume
Final concentration
H 2 O
23.4 μL
–
PrimeSTAR Max Premix (2Â)
2 5 μL
1 Â
Forward primer (100 pmol/μL)
0.3 μL
0.6 μM
Reverse primer (100 pmol/μL)
0.3 μL
0.6 μM
Template (5 ng/μL)
1 μL
0.1 ng DNA/μL
Total
50 μL
Table 6
PCR primer–template combinations
PCR product
Forward
primer
Reverse
primer
Template
Name
Size
Fab L
700 bp F1
R1
Native or synthetic cDNA encoding Fab light
chain
iRATmC 980 bp IRAT70-F
IRAT70-R
pFBgp67-iRATmC
Fab H
700 bp F2
R2
Native or synthetic cDNA encoding Fab heavy
chain
VEC-FB 4800 bp FB-F1
gp67-R
pFBgp67-iRATmC
Production of Recombinant Antibody Fragments via the iRAT system
93