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Cloning of big inserts


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#1 EvelyneR



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Posted 23 January 2017 - 12:46 AM

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?



#2 bob1


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Posted 23 January 2017 - 08:14 AM

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.

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