NS1 and control plasmids were isolated using Qiagen Midiprep plasmid isolation kit. Bacterial culture grown overnight was harvested by centrifuging at 1900g for 20 min at 4 °C. The supernatant was discarded and the bacterial pellet was dissolved in 4 mL of buffer P1 and mixed properly. To this, 4 mL of buffer P2 was mixed thoroughly by vigorously inverting the tube four to six times and incubated at room temperature for 5 min. Finally, 4 mL of buffer P3 was mixed thoroughly by vigorously inverting the tube four to six times and incubated on ice for 15 min. The tube was centrifuged at 1900g for 30 min at 4 °C. The Qiagen midi column was equilibrated with 4 mL of buffer QBT and bacterial supernatant was loaded onto the column, and the column was allowed to empty by gravity flow. The plasmid was eluted from the column by using QF buffer. Isopropanol was used to precipitate the plasmid. Finally, plasmid was dissolved in autoclaved water. Purity and quantification of plasmid was checked using Nanodrop by measuring the absorbance ratio at 260/280 nm.
Midiprep plasmid isolation kit
Manufactured by Qiagen
The Midiprep plasmid isolation kit is a laboratory product designed for the purification of plasmid DNA from bacterial cultures. It provides a reliable and efficient method for the extraction and isolation of plasmid DNA on a medium scale.
Automatically generated - may contain errors
3 protocols using midiprep plasmid isolation kit
1
Plasmid Isolation from Bacterial Cultures
2
Generating CRISPR-Cas9 Knockout Cell Lines
3
Antibody Library Sequencing Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Bacterial pools from panning output of AgSC and TBC libraries against antigen D were used to amplify the VH genes for NGS. The glycerol stocks of bacteria infected with the panning outputs from AgSC, and TBC libraries (1.5 ml each) were used to extract the phagemids with a midi-prep plasmid isolation kit (Qiagen, catalog no. 12943). VH genes were amplified with primers specific for the signal peptide and CH1 regions. Briefly, in a 50 µl reaction volume, 1 µl of phagemid was mixed with 1 µl 10 mM dNTPs (Invitrogen, catalog no. 18427088), 1 µl each of 10 µM forward and reverse primers, 0.5 µl of Q5 hot-start polymerase (NEB, catalog no. M0493L), 10 µl Q5 reaction buffer and 35.5 µl nuclease-free water; PCR was performed at 98 °C for 30 sec (initial denaturation) followed by 25 cycles of 98 °C for 10 sec (cycle denaturation), 72 °C for 30 sec (primer annealing), 72 °C for 30 sec (primer extension), and final extension at 72 °C for 5 min. The amplified VH gene fragments were separated on a 1% agarose gel followed by gel purification (Macherey–Nagel, catalog no. 740609). The purified VH gene fragments were sequenced by the Amplicon-EZ service (Genewiz), which provides a sequencing depth of 50,000 paired reads with 2 × 250 bp length through an Illumina Mi-Seq sequencer.
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