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.

Dear Kenneth,

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.
Good Luck,
Roger Anderson
Publisher Anderson's Timesaving Comparative Guide ; Restriction Enzyme Edition

 



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