cloning in a long vector (~10kb) without sequence changes - (May/31/2006 )

hi, i found this website by accident, i hope someone can help me. (i'm german so my english might not be too perfect)

i try to clone a 1.6kb insert into a about ten kb plasmid.

plasmid was cut with Stu(blunt) and BstEII (sticky) , there are no others restriction sites available.
Fragment were PCR-generated (5'end with the ongoing sequence for the Stu-cut) and 3' end was digested with BstEII.
each RE was used in a high concentration over Night to guarentee definite cutting, and vector and inserts were gel purified (sea-kem agarose 0,5%) and extracted by freeze-squeeze and Phenol/chlorform purified and EtOH concentrated.

the problem is: i do ligate a sticky and a blunt end.. i've tried several protocols (1h up to 8h sticky conditions : 4°C, usind NEB or roche T4 ligase using 100ng of vector and different insert concentrations and overnight with blunt conditions: adding 15% f.c. PEG, water, buffer and new ligase to the reaction mix.) cell were electroporated (yes they are very competent, even for long vectors) and selected on amp plates.
i even tried a different way by digesting the vector with mungbean to create only blunt end(insert was of course then not BstEII digested) but that didn't work under blunt conditions..

i'm now in the end of my diploma phase and getting quite nervous..
i will try to use klenow to create blunt ended vector and a new PCR-fragment (because my sequence may NOT be altered, cause i work IN a gene, trying to exchange some bases -- PCR mutagenesis without a kit didn't work as well)

i hope this is quite understandable, if anyone has a great blunt-sticky ligation protocol or another good idea, please let me know, i'm getting desperate and loosind my enthusiasm on labortory work

davewww

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might be a silly question but : do you dephosphorylate your vector?
I know that generally ligation is done on two REdigested fragments. So there is only one phosphate at the end. Does triphosphate is ok for ligation?...
dou you add PEG in your ligation?

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for all cases i woulnd't expect religation cause the ends are not compatible. I've tried with and without CIP, it didn't work either way.

For the sticky part, i don't add PEG, but then i use 15% f.c. as it is typical for blunt ligation
(i hoped my ligation would work that way: sticky condition leading to sticky ligation for 1 up to 8h ; then shifting it to blunt condition allowing the now half-ligated blunt longer fragments to "religate".
I of course used a control, which didn't show any colonies at all so i shouldn't have uncut vectors and/or religated vector which weren't completely digested.

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