problems with midiprep
Posted 04 July 2012 - 05:32 AM
i am having 5 DNA constructs. they have same vector backbone. the insert is different by means of tag positions. these constructs were sequenced after miniprep and they were all good. i cultured the bacteria in 50 mL culture volume with slection resistance. then i tried to do midiprep using quiagen kit (a filter to get rid of the cell debris, column to bind DNA, and vaccum pump to wash). I did same treatment to all 5 constructs and i got good yield in 3 of them and the other 2 were poor. I repeated the experiment for those 2 constructs and the yield for those 2 particular DNA were still poor. If anybody have such experiences before or have any suggestion, is more than welcome.
Posted 04 July 2012 - 06:50 AM
Posted 04 July 2012 - 07:27 AM
well, i actually did not measure the OD! but the pellet size for all of them (this is very much the usual pellet size which i had during my previous experiments with several other constructs) were quite similar.
right now i had the idea of using less volume of bacterial culture (35mL) and to run a gel after adding the neutralizing buffer!
any other suggestions are more than welcome. thnx
Posted 04 July 2012 - 12:48 PM