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?
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)
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.
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??