cloning with Ecl XI cut fragment.
Roger F. Anderson
rander at cco.caltech.edu
Wed Nov 23 18:26:51 EST 1994
you at somehost.somedomain (Kenneth Y. Ilio) writes:
>i have been trying to clone a 1.6 kb fragment cut with ecl xi and bgl II
>enzyme into a xba i cut 3.2 kb expression vector after klenowing.
>everything that i have tried has failed. these failed attempts include
>ligating at both room temp and 14 C. i have done the exact same things
>with other fragments but cut with different enzymes. i think the problem
>is due to ecl xi although the manufacturers says that the enzyme is ok.
>another strategy the i am using at present is cutting the original
>fragment out with nco i/bgl ii. my concern is that nco i removes the
>kozak sequence. thus, these are my questions?
>1) has anyone used ecl xi?
>2) can anyone suggest an alternative to my cloning techniques?
>3) is there any vector out there that will allow me to clone an nco i cut
>fragment and restore the kozak sequence?
>i will appreciate any suggestions. thank you.
I am still a little unsure of your set-up but you should be able to
find several vectors with Eag I (A MUCH less expensive iso.) and Xba I
sites in them. Remember Eag I is the core of the Not I site. The over
hang is compatible so you can cut the vector with Not I if there is
a problem there.
Again some information about the vector and such would help.
Publisher Anderson's Timesaving Comparative Guide ; Restriction Enzyme Edition
More information about the Methods