Mini-Prep Blues

the End jgraham at bronze.ucs.indiana.edu
Sat Jul 9 13:23:16 EST 1994


You may want to try alkaline lysis again, but use ammonium acetate as your 
third solution rather than sodium or potassium acetate*. For cells
resuspended in 200 ul and denatured with 400 ul of SDS/NaOH, add
300 ul of 7.5 ammonium acetate at the dissolved pH. This change 
dramatically increase the number of enzymes which cut well in my
experience.

For super-clean sequencing templates, add RNAase to the supernatant
follwing this step, precipitate with 400 ul of isopropanol, and then 
resuspend the pellet in 2 M ammonium acetate, spin out the  "rocks"
and reprecipitate**. 

"We don't need no steenkin phenol or mystery resins".

Jim
J. E. Graham 
Biology and Chemistry Departments 
Indiana University Bloomington 


* Morelle, G. Focus 11:1 p.7
** Lee and Rasheed. 1990. Biotechniques 9(6) p.676 



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