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Crude lysis Plasmid isolation

plasmid isolation

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#1 D.Luffy

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Posted 04 May 2018 - 04:49 AM

Hey guys, Can you explain me the mechanism of crude lysis (boiling lysis) of plasmid isolation?How it affects the plasmid and when this method can be used?



#2 OldCloner

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Posted 08 June 2018 - 12:08 PM

Sure.  Rapid boiling minipreps are fine for getting small, high-copy number plasmids from nice clean E. coli host strains like DH5-alpha, which does not produce exonucleases, and when all you need to do with the product is cut it with restriction enzymes, run it on a gel, gel-purify an insert, etc.  These preps are NOT, repeat NOT appropriate for Sanger sequencing by capillary electrophoresis, because there is always a bit of left-over cell debris glitch, and this can block and damage the capillary, and the sequencing people will not be happy with you. You can use rapid boiled plasmids for screening, but always make a good spin-column prep if you want to sequence it.  These days the commercial spin column preps are good and cheap and there is no longer any real need to use rapid boilers.

 

The process obviously lyses the cell and denatures the proteins and the larger (chromosomal) DNAs, which are then pelleted in ball of goop and removed (mostly) with a toothpick, leaving behind the smaller, tougher supercoiled plasmid DNAs to be recovered from the aqueous portion.  If you are doing large plasmids (over maybe 10 kb), or low-copy plasmids, it can damage the plasmids too much, so it would not be the best method for those.







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