'Cracking'

S.Nakayama, NIAR shignak at SS.ABR.AFFRC.GO.JP
Wed Jun 9 23:19:42 EST 1999


Hi Robert,

At 6:20 PM 99.6.8, Dr. Robert Gay wrote:
> We're looking for a protocol for the rapid screening of plasmid DNA
> directly from transformed E.coli colonies (avoiding the overnight liquid
> culture step).
>
> It's a protocol you may know as 'Cracking'.  We had such a protocol, once
> upon a time, but have  lost it !

We also faced same problem and searched the 'Cracking' protocol. But we can
not find. So, we employed PCR method for insert check.
1) Same number of colonies, PCR reaction mixtures each 10-15 uL including
Taq, primers (M13 or T3/T7/SP6...), dNTPs and buffers are prepared.
2) Colonies are picked by toothpick, put on new plates (numbered) and
resuspend in mixture (shake a few times). Plates are cultured at 37C.
3) Mixtures are heated at 95C for 10 min, cycled for 20-25 times at
95-50-72C and checked by gel electrophosis.
4) Confirmed colonies are cultured from plate (step 2) in liquid medium and
miniprep.
This works well, easy for us (cytogenetist as me) and reducing time (and
money).
We're checking insert size in the plasmid, PCR products are almost 200
bases longer than inserted sizes.

Someone tried the kit of Gibco BRL?
We saw the three color tubes in the catalog.

Best wishes,

Shigeki Nakayama

!(^_^)!|(-_^)||(-_-)|m(_ _)m<(   )>w( - )w|(-_-)||(^_-)|!(^_^)!
                                               Shigeki Nakayama
                                         shignak at chromosome.net
                                      http://www.chromosome.net
!(^_^)!|(^_-)||(-_-)|w( - )w<(   )>m(_ _)m|(-_-)||(-_^)|!(^_^)!





More information about the Methods mailing list