i digested a plasmid to extract my sequence of interest however i always get a faint band which means low dna. I tried increasing amount of dna digested to 2 ug but the band of interest is still faint but the top band/back bone is very bright and big.. i suspect there wasn't enough digestion? i used a 30 uL reaction volume for 2 ug DNA and 2 hour incubation period and NEB enzyme where am i going wrong? i need a lot of insert DNA
Another prob when i digest my vector with the backbone my yields aren't to great either but they sufficient and then i do dephosphorylation and after column clean up i have lost 50% of my DNA how do i reduce the amount of lean up steps because the process was: digest, gel extraction that included column purification, and then dephosphorylation followed by another column purification step.... in the past i did dephosphorylation directly after digestion because it was a thermo scientific product that would work in thermo scientific enzyme buffer but now we use NEB enzyme and i dint think thermo dephosphorylation kit works in NEB buffer.
Edited by KhaleesiDany, 23 January 2015 - 01:29 AM.