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Bacterial lysis buffer


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#1 rasi

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Posted 17 January 2011 - 08:45 PM

Hi everyone,

I just wanted to know if there was a way for lysing E.coli bacteria without using lysozyme?

The lysis buffer we use has 10mM tris-HCl, 1mM EDTA, Triton X-100, 150 mM NaCl and SDS.
However previously they used to add lysozyme before sonicating for 7 mins at 40-40 pulses.

So would the bacteria still get lysed with this method even if I dont added lysozyme?

Thanks in advance!

#2 protolder

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Posted 17 January 2011 - 10:40 PM

Hola, only sonicating without any detergent you broke bacteria, if any detergent is added you solubilize some of the insoluble proteins as the integral of membrane.These sonication conditions are for the pellet of 1 liter or more,arenīt they?. Buena suerte

#3 rasi

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Posted 19 January 2011 - 04:08 PM

Hola, only sonicating without any detergent you broke bacteria, if any detergent is added you solubilize some of the insoluble proteins as the integral of membrane.These sonication conditions are for the pellet of 1 liter or more,arenīt they?. Buena suerte


Actually we use these sonicating conditions for E.coli grow in about 200ml of broth and after they reach an OD of 0.6 I centrifuge it and resuspend it in about 3ml of lysis buffer.

SO just to clarify you reply, I should be able to lysis the bacteria with just the sonicating process?

#4 protolder

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Posted 19 January 2011 - 11:03 PM

Hola, yes you can. I donīt know what is your sonicator and what the frecuency, but for the pellet of 200ml at OD 0.6 , 3 pulses of 30s at medium power in ice bath in a volume of 5ml will be enough, but look under microscope the bacteria before and after sonication, if after sonication you can see whole bacteria, try more pulses or power. Buene suerte




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