too low DNA conc. after ligation..help! - (Jun/20/2005 )
i have problem with ligation and transformation of my knockout construct into lactis. finally after 5 months of "try again" cloning method, i got one colony on my plate (M17, EM marker 1 ug/ml)
when i digest it with Nco1/Xba1, i have two bands that supposed to be righ (my vector 5.2kb, KO insert 632bp) but with a very low amount of insert DNA. i cultured 10ml and did minipred on them, but still not that much of insert DNA recovery.
could anyone tell me what should i do to increase the DNA conc.?
and what arre the reasons for such poor efficiency of tranformation (i electrophorated lactis, gram +), 40ul competent cells, 2ul insert)
i think that the relative amount of bacterias and DNA in the transformation is not affecting the quantity of miniprep at the end. You have surely a low copy plasmid.
do you use a alcaline lysis method or a kit to do your minipreps? is your bacterial lysis ok? do you mix your samples at each time of miniprep?
Usually whien i do a alcaline lysis miniprep (from 2ml bacterias prep) i check about 1/6 on gel and it's ok. When i use promega miniprep (13ml starting) i check 1/50 and it's ok...