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failure in miniprep

plasmid extarction

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#1 LadyA2A



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Posted 07 December 2011 - 01:45 AM

Hi every one,
i'm trying to extract pGEX from E. coli (BL21) ... i'm using an alkaline lysis method ... but it did not work.. i see nothing on agarose gel.. only some genomic DNA broken down..
If anyone could help me then it will be very kind of you.

#2 phage434



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Posted 07 December 2011 - 06:07 AM

Extraction from BL21 strains is much more challenging than from cloning friendly strains such as DH5a, due to the presence of endonuclease. In general you want to avoid needing to do this, and work instead with endA- strains. Rapid lysis in the presence of denaturing reagents will help. You could try quickly moving from chilled P2 (SDS and NaOH) to chilled N3 (Guanididium HCl) if you are doing a Qiagen style prep, or you could do freeze/thaw multiple times or mechanical disruption (sonicator, bead beater) directly into N3 followed by column cleanup.


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