21. Run the PCR according to the following parameters: (1) 2 min
at 94
C; (2) 25 cycles of a sequence composed of 30 s at
94
C/1 min at 55
C/1 min at 72
C; and (3) 5 min at 72
C.
22. Load 10 μL of each colony PCR product directly onto a 1%
agarose gel, alongside an appropriate DNA ladder. After ethidium bromide staining, observe the gel by UV illumination.
The expected size of the colony PCR product is approximately
2500 bp.
23. Select several positive colonies, and use them to inoculate 6 mL
of LB/ampicillin medium. Shake the cultures overnight at
37
C and 250 rpm.
24. Day 3: Add 1 mL of each saturated overnight culture to 0.5 mL
of 50% glycerol in a 2-mL screw top tube or cryovial and gently
mix. Freeze the glycerol stock tube at À80
C. The stock is now
stable for years, as long as it is kept at À80
C.
25. Extract and purify the plasmid DNA from the remaining 5 mL
of each culture using a QIAquick spin miniprep kit, according
to the manufacturer’s instructions. Measure the DNA concentration using a NanoDrop spectrophotometer.
26. Using the vector primers, SFseq-F and SFseq-R, perform
sequence analysis to confirm that the nucleotide sequence of
the plasmids is correct.
27. Day 4: Chill 1 μL of the positive pFBgp67-iRATmC-based
entry plasmid in a 1.5-mL centrifuge tube.
28. Thaw the E. coli DH10Bac competent cells (10 μL) on ice.
29. Add 10 μL of DH10Bac competent cells to the plasmid DNA.
Mix gently by pipetting up and down to mix the cells and
DNA. Do not vortex.
30. Place the mixture on ice for 30 min.
31. Heat shock at 42
C for 45 s.
32. Place the mixture on ice for 2 min.
33. Add 90 μL of pre-warmed SOC medium to the tube.
34. Incubate the tube at 37
C for 4 h with vigorous shaking
(250 rpm).
35. Dilute the transformation culture with SOC medium as appropriate, and then spread 100 μL of cells onto an LB/KTGIB
agar plate.
36. Incubate the plate for 2 days at 37
C.
37. Day 6: Pick several white colonies, and use them to inoculate
6 mL of LB/KTG medium. Shake the cultures overnight at
37
C and 250 rpm.
Production of Recombinant Antibody Fragments via the iRAT system
95
at 94
C; (2) 25 cycles of a sequence composed of 30 s at
94
C/1 min at 55
C/1 min at 72
C; and (3) 5 min at 72
C.
22. Load 10 μL of each colony PCR product directly onto a 1%
agarose gel, alongside an appropriate DNA ladder. After ethidium bromide staining, observe the gel by UV illumination.
The expected size of the colony PCR product is approximately
2500 bp.
23. Select several positive colonies, and use them to inoculate 6 mL
of LB/ampicillin medium. Shake the cultures overnight at
37
C and 250 rpm.
24. Day 3: Add 1 mL of each saturated overnight culture to 0.5 mL
of 50% glycerol in a 2-mL screw top tube or cryovial and gently
mix. Freeze the glycerol stock tube at À80
C. The stock is now
stable for years, as long as it is kept at À80
C.
25. Extract and purify the plasmid DNA from the remaining 5 mL
of each culture using a QIAquick spin miniprep kit, according
to the manufacturer’s instructions. Measure the DNA concentration using a NanoDrop spectrophotometer.
26. Using the vector primers, SFseq-F and SFseq-R, perform
sequence analysis to confirm that the nucleotide sequence of
the plasmids is correct.
27. Day 4: Chill 1 μL of the positive pFBgp67-iRATmC-based
entry plasmid in a 1.5-mL centrifuge tube.
28. Thaw the E. coli DH10Bac competent cells (10 μL) on ice.
29. Add 10 μL of DH10Bac competent cells to the plasmid DNA.
Mix gently by pipetting up and down to mix the cells and
DNA. Do not vortex.
30. Place the mixture on ice for 30 min.
31. Heat shock at 42
C for 45 s.
32. Place the mixture on ice for 2 min.
33. Add 90 μL of pre-warmed SOC medium to the tube.
34. Incubate the tube at 37
C for 4 h with vigorous shaking
(250 rpm).
35. Dilute the transformation culture with SOC medium as appropriate, and then spread 100 μL of cells onto an LB/KTGIB
agar plate.
36. Incubate the plate for 2 days at 37
C.
37. Day 6: Pick several white colonies, and use them to inoculate
6 mL of LB/KTG medium. Shake the cultures overnight at
37
C and 250 rpm.
Production of Recombinant Antibody Fragments via the iRAT system
95