PCR amplification of the cloning inserts was done
using Q5 High-Fidelity 2X Master Mix (NEB, Ipswich, MA, catalogue
no. M0492L). Twenty μL reactions were prepared by dispensing
1 μL of each 10 μM reverse primer into the wells of a
96-well PCR plate using the Echo liquid handler (Beckman Coulter,
Brea, CA). A Mastermix consisting of polymerase premix, plasmid DNA
template (pBbB6c, 5–10 ng per reaction), and the single 10
μM forward primer was prepared and dispensed using the FeliX
liquid handler (Analytik Jena, Jena, Germany) or electronic multichannel
pipet. Reactions were run using Touchdown PCR or standard PCR cycling
methods in C1000 thermal cyclers (Bio-Rad, Hercules, CA). Capillary
electrophoresis of PCR products was performed using the ZAG DNA Analyzer
system (Agilent Technologies, Santa Clara, CA). Two μL of each
PCR reaction was electrophoresed using the ZAG 130 dsDNA Kit (75–20 000
bp) or ZAG 110 dsDNA Kit (35–5000 bp) (Agilent Technologies,
catalogue no. ZAG-110–5000; ZAG-130–5000). ZAG sample
plates were prepared using the Sciclone G3 liquid handler (PerkinElmer,
Waltham, MA). ProSize Data Analysis Software (Agilent Technologies)
was used to generate gel images from the sample chromatograms, and
amplicon sizes were estimated by reference to the upper and lower
DNA markers spiked into each sample and a DNA ladder run in well H12
of each sample plate.
using Q5 High-Fidelity 2X Master Mix (NEB, Ipswich, MA, catalogue
no. M0492L). Twenty μL reactions were prepared by dispensing
1 μL of each 10 μM reverse primer into the wells of a
96-well PCR plate using the Echo liquid handler (Beckman Coulter,
Brea, CA). A Mastermix consisting of polymerase premix, plasmid DNA
template (pBbB6c, 5–10 ng per reaction), and the single 10
μM forward primer was prepared and dispensed using the FeliX
liquid handler (Analytik Jena, Jena, Germany) or electronic multichannel
pipet. Reactions were run using Touchdown PCR or standard PCR cycling
methods in C1000 thermal cyclers (Bio-Rad, Hercules, CA). Capillary
electrophoresis of PCR products was performed using the ZAG DNA Analyzer
system (Agilent Technologies, Santa Clara, CA). Two μL of each
PCR reaction was electrophoresed using the ZAG 130 dsDNA Kit (75–20 000
bp) or ZAG 110 dsDNA Kit (35–5000 bp) (Agilent Technologies,
catalogue no. ZAG-110–5000; ZAG-130–5000). ZAG sample
plates were prepared using the Sciclone G3 liquid handler (PerkinElmer,
Waltham, MA). ProSize Data Analysis Software (Agilent Technologies)
was used to generate gel images from the sample chromatograms, and
amplicon sizes were estimated by reference to the upper and lower
DNA markers spiked into each sample and a DNA ladder run in well H12
of each sample plate.