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Isolation of Cosmids using Eppendorf's Perfectprep Plasmid ,,, Mini Kit

easy and efficient way of isolating cosmids from bacterial culture without shearing or significant loss of product due to the binding properties of cosmids.

This article shows the capability of the Perfectprep Plasmid Mini Kit in isolating several cosmid constructions for screening purposes. A specially designed vector designated pLorist6Xh [3] was used in this experiment for screening of the Neurospora crassa genome. Restriction analysis of the pLorist6Xh vector isolated from the Perfectprep Plasmid Mini Kit was performed for analysis of DNA. The procedure shows the ability of the Perfectprep Mini Kit to isolate the pLorist6Xh vector containing inserts ranging from 1842kb.

Materials and Methods

  • Bacterial Culture
    DH5a was used as the host strain for cosmid insert pLorist6Xh. Selected colonies were inoculated in 3 mls of LB with kanamaycin at a final concentration of 50 mg/ml. The cultures were placed in 14 ml snap cap tubes and allowed to grow at 300 rpm and 37C for 1416 hours, optimal growth will give OD 600 readings of an 1.52.0.
  • Purification
    Cells were harvested at 4000 rpms for 6 minutes at room temperature in a 5810 centrifuge with rotor A-4-62. Removing supernant, harvested cells were resuspended in 100 ml of solution 1 (see Protocol below). Vortexing of sample allowed for complete resuspension. Aliquots of 1 ml samples were transferred to 1.5 ml tubes. The purification process was completed occurding to the Perfectprep Mini protocol below. Elution was carried out with 70 ml of elution buffer heated to approximately 60C to elute cosmid DNA.
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