Cloning of big inserts - (Jan/23/2017 )

Hi all,

At the moment I need to clone big inserts in an expression vector (pcDNA3.1+). The vector size is 5.4 kb, and my inserts range from 5.1 to 4.2 kb, which is already difficult, but I also have to cut with different enzymes. The pcDNA3.1+ vector is cut with EcoRI ans AscI (sequential digest, starting with EcoRI), and the inserts are cut with MfeI and MluI (also sequential digest, starting with MfeI). I've already used a 1:1, a 1:3, and a 1:5 vector:insert ratio, using 20 or 30 ng of pcDNA3.1+. I use T4 DNA ligase (high concentration), and ligated for 45 minutes at 22°C. So far I've had no positive colonies.

Does anybody have any idea about how to make this work?

Cheers

Your inserts do not have compatible ends with your vector as the restriction enzymes are producing different overhanging sequences.

If you can't find compatible ends in the sequence, you can either do an adapter ligation or PCR the sequence with the desired ends added to the primers. You could also fill in the overhangs and then do a blunt ligation, but this would remove any directionality to the insertion, which is probably what you are aiming for given that you are inserting into pcDNA3.

There is also the possibility of putting your insert into something like pUC first then into the pcDNA plasmid, but you will still run into the problem of compatible ends.

You should also run transformation controls to ensure that your bacteria are competent.

It seems to me that EvelyneR's plan should work- the termini of EcoRI and MfeI are compatible and the termini of AscI and MluI are also compatible.  However, glancing at a map of pcDNA3.1+  I've pulled up from the internet, I do not see a AscI site in the multiple cloning region of the vector. There are several versions of pcDNA vectors and you should make sure you have the one that matches your map if you plan on using AscI. (It's possible not everything is listed on the map I pulled up, so just double check) Especially if you got the vector from someone other than the supplier (Invitrogen).  If that site is not there, (Or if it in the backbone of the vector rather than the MCS) it would explain the whole failure.  BTW, don't be scared by 5.1 kb inserts; to me that's not very big at all!

You can try cutting the vector with AscI alone and run it on a gel to see if it cuts.  If it doesn't-there's your problem.