methods-revision3 - Polina Pomerants Methods Construction...

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Polina Pomerants Methods Construction of plasmid M-CSF-encoding plasmid was constructed by inserting a PCR-amplified cDNA nucleotide sequence of simian immunodeficiency virus (SIVmac251) into the 72 bp polylinker site of the previously purified 3107 bp-Escherichia coli (E. coli) transcription vector pSPT19 between SP6 and T7 RNA polymerase promoters in the region of base pairs 16 and 36 (Figures 1, 2) (Roche Applied Science). The SIVmac251 sequence was taken from the GenBank database (accession no. M19499) and included the entire gp120 and gp41 regions of env and nef gene (Cite). Purification of plasmid DNA A construct of DNA-containing plasmid was isolated from (E. coli) (Qiagen plasmid maxi kit). In order to survive and grow, a single, well isolated colony of ampicillin resistant E. coli containing the plasmid, was inoculated with a starter culture of lysogeny broth (LB) media containing ampicillin. The bacteria were incubated overnight at 37°C with vigorous shaking (~ 300 rpm). Bacterial cells were harvested by centrifugation at 6,000 x g at 4 °C. The resultant bacterial pellets were resuspended in buffer P1 [50 mM Tris•Cl; pH 8.0,10 mM EDTA; 100 µg/ml RNase A], which ensured that any pieces of cellular RNA in the plasmid would be degraded (Qiagen). Pellets were lysed with buffer P2 [200 mM NaOH; 1% SDS (w/v)], and the lysate was neutralized with chilled buffer P3 [3.0 M potassium acetate, pH 5.5] (Qiagen). Pellets were
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subsequently centrifuged at 20,000 x g at 4 °C and supernatants containing the plasmid DNA were promptly removed. The supernatant was bound to the gravity-flow column, was washed with Buffer QC [1.0 M
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methods-revision3 - Polina Pomerants Methods Construction...

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