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Maxi plasmid preparation - (Jan/04/2008 )

I prepared plasmid from DH5alpha using the classic alkaloid lysis method (non-kit);
After adding solution I/II/III,centrifugation, 0.6 volume of iso-propyl alcohol,precipitation,washing using 70% alcohol, the protocol said "dissove the precipitates using 3 ml TE buffer and add the same volume of LiCl...."
actually, the TE buffer can not dissove the precipitates completely..
Any one can tell me why?

-glcui-

The TE isn't able to disolve the pellet easily due to heavy protein contamination. This is normal. Just do your best to break the pellet up using a pipette or dropper (if you are working on large BACs). The protein will be cleaned up using phenol/chloroform extraction step which comes after LiCl percipitation step (which removes RNA)

-perneseblue-

well if i remember good LiCl if for removing RNAs?.
you can heat your pellet at 55° for 5 to 15' vortexing regulary (better are agitating heating blocks).
Then you can proteinase K treat your sample or add the LiCl.

-fred_33-

i routinely do maxi prep using classic alkaline lysis mehtod but never faced this problem. 3 ml TE is enough to dissolve the ppt i got after ehtanol wash.
do you wash your cells with STE buffer before adding sol I??

-T. reesei-