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

erfect Prep Mini Protocol:
  1. Pellet 1 to 3 ml bacterial culture by centrifuging for 20 seconds. Remove supernatant by aspiration or decantation.
  2. Add 100 l Solution I to the pellet and completely resuspend cells by vigorous vortexing.
  3. Add 100 l Solution II and mix well by repeated gentle inversion.
  4. Add 100 l Solution III and mix well by repeated vigorous inversion.
  5. Centrifuge lysate for 30 seconds and transfer supernatant to a spincolumn.
  6. Add 450 l well-mixed DNA Binding Matrix Suspension; mix well.
  7. Centrifuge for 30 seconds. Decant filtrate from collection tube.
  8. Add 400 l Diluted Purification Solution to the spin column; shake briefly. Centrifuge 60 seconds.
  9. Decant filtrate and place spin column back in the collection tube. Spin for 60 seconds.
  10. Transfer spin column to a fresh collection tube. Add 50 to 70 l of Elution Buffer to DNA Binding Matrix. Vortex briefly.
Centrifuge for 60 seconds, Discard used spin column and store eluted plasmid DNA at either +4C or 20C.

Analysis

Restriction digests were carried out to a final volume of 15 l using BamHI [4] and 7.5 l of each purified sample. The reactions were incubated at 37C for 1 hour. Electrophoretic analysis was carried out using all 15 ls of restriction digests loaded on a 0.7% Amresco Agarose III gel and run at 100 V for 2.5 hours. Two DNA standards were also loaded on the gel; l HindIII and 1 kb ladder [5].

Results and Discussion:

The ability of the Perfectprep Plasmid Mini to isolate large size cosmids is shown by the restriction digest
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Source:


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