Insert magically shortening??? - Cloning issues (Jun/09/2011 )

Hey everyone,

I am trying to clone a 12kb insert into a pBS vector (~4kb) using In-Fusion cloning which uses 15bp homologous ends instead of TA cloning. I linearized the vector with EcoRV which is a blunt end digestion enzyme and gel purify it. I am also using Takara's LAPCR to amplify the 11kb insert and gel purify it as well. I go through the ligation, trans, and plating, however when I screen the colonies I get some very weird results.

First, using digestion enzymes to linearize the vector and vizualize it on a gel, I only get a length of around 7-8 kb which would mean that my insert is only ~3-4kb instead of 12kb. I"m not sure why this is happening, or how this would even happen. Also, to confuse things even more, I have been screening these inserts by doing a basic PCR using primers that target the 3', 5' and middle section of the insert and they all amplify at the correct lengths which would suggest that the entire insert is present in the vector. But the digestion says otherwise.

Any suggestions/comments/experiences?

Thanks!

Do the three PCR cover the entire 12kb insert? Do the three PCR reaction amplify the entire 12kb insert?

PCR only says a specific segment is present. It says nothing but the integrity of the entire DNA segment. If the 3 PCR reaction do not amplify the entire insert, it is then possible that a segment of the insert is missing.

It is also possible that the EcoRV enzyme cuts the insert more than once... maybe twice giving two 6kb fragments.

I would suggest trying a few different restriction enzyme that cuts the insert 2-3 times. See if you get the expected number of DNA bands of the correct size.

That way you can find out if a segment is missing from the insert.

The PCRs only amplify small segments of the insert. The idea was that with a gel purified 12kb insert, there shouldn't be anyway to modify it once its in the cell so if we can amplify the 3' and 5' end, it should all be there. Do you think there are issues with that thought?

So I have used a few different enzymes to digest my recovered plasmid from the cells and all the fragments from each digestion add up to around 8kb instead of the 16kb (12kb insert+4kb vector) that I'm expecting.