Hi Shirin,

There are two kinds of plasmids. High copy plasmids in bacteria give high concentration after isolation however the other type low copy plasmids give low concentration. I do not know which plasmid you used but, I assume your plasmid is low copy. You have to increase the culture volume, after isolation, you can concentrate your plasmid with evoporation, in case you want to use high concentration of plasmid. Therefore, please check the plasmid whether low copy or not.

Best

Hi Shirin,

Please mention name of your source strain and antibiotics resistance genes present in the plasmid. I will suggest you a optimized protocol based on the information you provide.

Tips

1)Include antibiotic solution in the culture broth to increase plasmid copy number

2)Minimize ethanol washing steps to increase the yield.

Yes Cigdem Ozen  is right.

Dear Shirin,

Plasmid you are using can be low copy plasmid. So increase in the culture volume will give you better yield. Another reason can be, if you are isolating plasmid from Agrobacterium strain rather than E.coli than you grow it for longer time to get enough plasmid for sequencing.

Hi ,

thanks for your kind replies. I do use high copy number plasmids for this experiment, also, i have tried growing in bigger volumes than usual and concentrating the plasmid after isolation but it still didn't help much. because i have used this vector before and i never had such low yield with this plasmid!! 

more info:

host is E-coli DH5a, Vector is high copy number and resistance is Ampicillin.

by the way, i wanted to add that even I tried sending the bacterial culture to the sequencing company and they as well have a hard time with isolating the plasmid, regrowing the culture so many times and at the end they report that sorry we could not isolate the plasmid of ur bacterial culture, hence we don't charge you :/