Do I need to change any protocol, E. coli strain or media I am using now to change to pSpark DNA cloning systems?
A7. No. Just set up a ligation according to catalogue Section 2.3.1 (or Quick Protocol for Advanced Users), incubate ligation up to 60 minutes at 22C (or overnight if needed) and follow all the regular protocols you are familiar with. Use a 3:1 to 5:1 (recommended) insert to vector molar ratio as starting point. That corresponds exactly to 6,7 ng of insert per kb for the recommended 5:1 insert-to-vector ratio. As a rule of thumb you can use 5ng per kb of insert to be cloned, that is, 2,5 ng, 5 ng or 10 ng if cloning an insert of 0,5 kb, 1kb or 2 kb. If cloning an unpurified PCR product use 1 uL of PCR directly for ligation.
