Prep of M13 RF DNA

Elmer M. Price, Ph.D. DCRCEP at mizzou1.missouri.edu
Thu Jun 23 14:35:41 EST 1994


In article <1994Jun22.170212.1 at hkucc.hku.hk>
vldnaseq at hkucc.hku.hk writes:
 
>
>I have an insert cloned in M13mp19 vector.  I have some difficulty in preparing
>the double stranded replicative form of the clone.  The culture was grown in
>2xYT until the O.D 600nm = 0.8 - 1.0 . The recombinant phage stock was added
>at m.o.i = 20 . After 15 mins, chloramphenicol was added (final conc. was
>15ug/ml).  The culture was harvested after 2 hr.  However, only 5 ug of ds DNA
>from 20 ml culture was obtained.  Anyone know the method of producing more ds
>DNA from M13 clone, please share your experience.
>
 
 
 
Hello-In the past, we have had very good success with a brute force approach.
We simply innoculate a culture of either 2xYT or some T broth 1:100 with an
overnight culture of bugs, usually DH5aF' and some infectious M13 (moi 1-10)
phage which contains our insert.  After 8 hours or so (even after an overnight
growth), the d.s. M13 is prepared as is any other double stranded plasmid.
We do not use chloramphenicol.  Also, the M13 is not lytic so attention to ODs
is not crucial.  The next step is to subclone the M13 insert into a more friend
ly vector!  Our yields are usually low (about 100-200ug per liter).
 
Hope this helps
 
Elmer
 



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