different digestion pattern for same clone
Posted 19 March 2009 - 09:31 AM
I picked some colonies and made mini-perp and also glycerol stock. When I did second mini-prep from direct glycerol stock (without streaking on plate), i havent got the samp digestion pattern as in my first attempt. what could be the reason?
1. Freez-thawing would have killed the clones? becos I have stored the glycerol stocks in -20 not -80 !!
2. Its that ok if I inoculate liquid culture directly from glycerol stock instead of streaking on plate ---> pick colony ---> miniperp?
Pls give ur suggestions
Posted 19 March 2009 - 03:55 PM
Innoculation from a stock is not good practice, 99% of the time it will work for what you want, but the 1% when it doesn't could lead you to have all sorts of errors in your experiments with what you thought was the correct plasmid/bacteria. Streaking and picking ensures that you have a clone from one bacterium, meaning that you can be more sure that what you are getting is the correct thing.
Posted 20 March 2009 - 03:59 AM
Posted 21 March 2009 - 10:42 PM
try to streak it and pick up colonies and if you still have problems, then you might have lost the plasmid.
Posted 24 March 2009 - 09:41 AM