cloning with Ecl XI cut fragment.

Kenneth Y. Ilio you at somehost.somedomain
Sat Nov 19 17:42:06 EST 1994


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. 



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