3 replies to this topic

[sb21]

Hi,

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.

Thanks

[phage434]

Did the cultures grow to similar ODs?  The two low concentration results may be from plasmids that are producing toxic products, inhibiting growth.

[sb21]

thanks for the response.
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

[Thapa]

All of your DNA constructs are not same. They may not equally pure. Transformation efficiecy and copy numbers may differ during transformation. Bacterial inoculam may differ and so the growth rate and OD. I use the same type of midi-preps and I usually get variable yeilds. Try new transformation, or streak and culture from your glycerol stock.