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cloning a large insert - (Feb/17/2011 )

hi all,
i am trying to clone a 7kb gene into a 6.2kb vector. I have tried different competent cell strains with no success. I also ran the ligation mix in an agarose gel and saw that ligation is working. How do i proceed with transforming this?
plss help. I am at my wit's end.


one is the competent cell efficiency, other is if you are using the right antibiotic resistance plates. I am sure you would haver checked these. I would try transformation with a commercial competent cell.


i tried electroporation as well, i get plasmids of size 9-10kb and random sizes of vector and insert when i restrict the plasmid. i always get colonies and they always have plasmids but never the correct size. where am i going wrong?


And you are sure your plamid doesnt contain multiple restriction sites?
And if so: you kept that in mind making the calculations?


ya, i checked for multiple sites in my plasmid, but there are none. i am sure i havent made an error in choosing the sites. i checked and asked a lot of others also to verify :(


is it a blunt or a sticky ligation? ...maybe you can give more details?