1. Day 1: Perform PCR amplifications of V L , the iRAT-MBP
segment, V H , and the pNY326 vector backbone (VEC-NY).
Add the appropriate components to four 0.2 mL PCR tubes as
indicated in Table 1. The PCR primer–template combinations
are listed in Table 2. The primers for the V L and V H domains
include 5
0 overhangs (27 bp) complementing the upstream and
downstream sequences to the pBIM2 vector junctions. Mix the
contents gently, and place the tubes in a thermal cycler.
2. Run the PCR according to the following parameters: (1) 2 min
at 95
C; (2) 30 cycles of a sequence composed of 10 s at
98
C/5 s at 56
C/5 s/kbp at 72
C; and (3) 10 min at 72
C.
3. Check the PCR products by loading an aliquot (5 μL) onto a
1% agarose gel with a DNA ladder in a separate lane. After
ethidium bromide staining, observe the gel by UV
illumination.
4. Purify the remaining PCR product (45 μL) using a Wizard SV
gel and PCR clean-up system, according to the manufacturer’s
instructions. Use the PCR products directly for purification.
DNA extraction from the agarose gel slice is not necessary.
Table 2
PCR primer template combination
PCR product
Forward primer Reverse primer Template
Name
Size
V L
360 bp
F1
R1
Native or synthetic cDNA encoding V L
iRAT-MBP 1300 bp BIM2-F
BIM2-R
pBIM2 vector
V H
380 bp
F2
R2
Native or synthetic cDNA encoding V H
VEC-NY
3400 bp pNY326-F
pNY326-R
pBIM2 vector
Table 1
Preparation of PCR tubes
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
Production of Recombinant Antibody Fragments via the iRAT system
85