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Using 10,000 rpm instead of 14,000 for DNA extraction with Promega kit

Centrifuge DNA extraction Promega

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#1 hercolanium

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Posted 07 January 2014 - 07:34 AM

Would it work in the extraction of DNA using Promega Wizard to use a centrifuge of 10,000 rpm instead of 13,000-16,000 but for longer time and if yes how much longer?



#2 bob1

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Posted 07 January 2014 - 11:51 AM

Which of the kits?  Which centrifuge?  IIRC the promega kits say to use "g" not rpm.  Note that rpm readings are useless unless you also know the diameter or radius of the rotor so that you can work out the relative centrifugal forces (RCF or "g").

 

Basically the answer is yes (with a few cautions), but the extension of time depends on what is being done by this step.  If the step is to just pass the solution through a filter and hence bind the DNA, or a washing step, then I would say just add a minute on to the time.  However, if it is a step where you are trying to remove all of the wash buffer before elution (alcohol residues inhibit downstream applications) then you may well need the higher force to get the buffer out against the capillary effect of the filter.  Steps to spin down precipitated protein before DNA extraction usually require the higher forces as the density of the protein is often pretty similar or lower than that of the buffer.  Steps to pellet DNA should also work, but may require quite a bit of extra time.



#3 phage434

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Posted 08 January 2014 - 07:05 AM

I think that this will work fine. As Bob1 indicated, the critical step you need care with is removal of ethanol from the wash in the final dry spin. An extended version of that spin (which I recommend in any case) will solve the problem.







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