problems in clone confirmation - (Jan/18/2008 )

Hi,
I am engineering a protein encoding gene that has provision of Bsu36Iand BbvCI sites in its Coding sequence, and it is already cloned in pQE30NST based expression vector. The insert I am cloning also has sticky BbvCI/Bsu36I sticky ends. However both the enzymes are reported to have less ligation efficinecy after cutting, less than 10% and 50% for BbvCI and Bsu36I respectively, as per NEB FAQ comments for those enzymes on ligation.

I set up ligation reaction, with 3:1, 6:1, and 9:1 insert to vector ratio, at standard final dna con of 10ng/ul, in a 20ul reaction vol. The size of vector is 6.66kb and insert is 2kb,but the ligation failed twice. Also, I did not dephosphorylate.

To compensate this lesser efficiency of religation with these ends, I increased the final dna concentration of my ligation reaction to 20ng/ul and 30ng/ul in a 20ul reaction volume, with a 1:1 insert to vector molar ratio, (as I was told with 2kb insert, 1:1 ratio should be fine). and also gave increments of ligase (used 1ul of 5x concentrated ligase (NEB).

I got clones, and isolated the plasmid and used appropriate restriction enzymes to confirm insert size, along with vector controls. Plasmid size of the tested clones were significantly less than the vector (6.6kb), although got good in yield, and to further confirm the size I did restriction digestion. With different enzymes the calculation came up so absurd, less than the vector size. Can any one has some explanation and alternative way without modifying the restriction sites?
the host is DH5-alpha, commercially available one.

Thanks in advance.

Looking at a map of your vector (http://www.proteinstrukturfabrik.de/tp03page/vectors/pqe30nst.pdf) I'm having trouble understanding why you chose the restriction enzymes that you did. Why not just use BamHI and NotI, e.g.? If the enzymes you are using are problematic, it might be faster in the long run to use some that are old stand-bys.

In terms of getting a unique restriction pattern, are you sure the restriction sites from your enzymes are not in your insert/vector anywhere? If so, this will explain the unusual pattern you are seeing.

Hope that helps!

Hi,

I would design primers that cover both the vector and the insert sequence and do a PCR. The would allow you to confirm the insert was inserted correctly and you could even send the PCR product off for sequencing to double check,

P