What is the best way to screen for recombinant DNA made with the AdenoVatorTM kit?
A. The restriction enzyme Bst XI gives a very good diagnostic pattern to screen recombinants with the AdenovatorTM system. The restriction fragments of the pAdenoVator-1 plasmid are as follows: 1- 11921 bp 2- 8237 bp* 3- 5251 bp 4- 4254 bp 5- 2379 bp 6- 1399 bp Fragment number 2 is the only one that will vary with the size of insert and from one transfer vector to another. With pAdenoVator-CMV5, it shifts up to about 11-12 kb (and higher with the insert) whereas with pShuttle, it shifts to approximately 10.5 kb. This band may sometimes be confused with band 1 but disappearance of band 2 is a good indication of positive recombinant. With pAdenoVator-CMV5-IRES-GFP/BFP, band 2 is cut in the IRES fragment to give a smaller shift of that band and an additional band at approximately 2 kb. This, in conjunction with the Pac I restriction pattern, makes identification of positive recombinants very straightforward.
