Biology Protocols

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Large Scale Prep for Very Large Plasmids and Cosmids

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  • Grow up the cells in 100ml volumes of LB.
  • Spin down the cells in 250ml buckets at 5K for 15 min in an RC-5B or equivalent.
  • Pour off the supernatant and carefully resuspend the cells in 6.6ml of 25% sucrose. Avoid any pellet by repeatedly pipetting.
  • Working on ice, add 1.34ml of fresh lysozyme solution (5mg/ml in 25% sucrose).
  • Leave on ice for 5 min.
  • Add 2.6ml of 0.25M EDTA.
  • Leave on ice for 5 min.
  • After 5 min remove a 1ml sample, add 1ml of Triton solution (1% Triton X-100 in 0.1MTris.HCl pH8) and gently mix. If the cells lyse - indicated by the solution becoming thick and viscous, proceed to the next step. If this does not occur leave the cells for another 5 min.
    (If the cells are very hard to lyse it may be worth carrying out the lysis at R/T or maybe adding Proteinase K).
  • Add 10.5ml of the Triton solution to each of the complete buckets carefully down the side of the bucket so that the Triton floats on the surface.
  • NB Add 1ml less to the bucket from which the sample was removed.
  • Carefully mix the two layers by repeatedly pipetting through an inverted 10ml pipette until lysis occurs.
  • Centrifuge at 10k for 30 min in a Sorval RC-5B or equivalent.
  • Carefully remove the cleared lysate from the viscous pellet - it is a bit like separating eggs.
  • Add a 1/2 volume of 30% PEG solution. Leave at room temperature for 30 min.
  • Spin down the precipitated nucleic acids (etc) in a 50ml centrifuge tube at 10K for 10 min in a Sorval RC-5B.
  • Resuspend the pellet in 11ml of TE buffer - you may need a whirlimixer.
  • Add 11.38g of CsCl, 0.22ml TE and 0.35ml of ethidium bromide. Fill a quick seal centrifuge tube and centrifuge in the L8 for 16-20 hr.
  • Remove the band containing the plasmid DNA (normally the upper band of two) and phenolise before ethanol precipitating the plasmid DNA.

Notes

Resuspending the DNA after ethanol precipitation may take a while, it is sometimes a good idea to leave pellet in the resuspension buffer for a while so as to maximise the yield.

Store large plasmids and cosmids in the fridge as they seem susceptible to damage when store in the freezer.

 

 


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