If you are using a low copy plasmid you can use a chloramphenicol protocol:

- mini culture on night

- maxi culture on the day

- on the evening you add your antibio, fresh medium and chloramphenicol: the bacteria growth will be blocked but not the synthesis of you're plasmid. it's working very well

1. Have you done other plasmid extraction from the kit purchased from Cosmo Genetich and showed good results?

2. Is your plasmid high copy number or low copy number?

3. You can increase the bacterial culture volume for plasmid isolation.

Just few generic suggestions:

-Use a bigger prep kit ---> midiprep/maxiprep and a bigger volume initial culture (you could resuspend the pellet in a smaller volume of nuclease free water and get a more concentrated DNA)

-If the plasmid is a low copy plasmid you should expect lower yield respect to an high copy plasmid. By the way you don't need micrograms of plasmid to get sequencing data, probably something gone wrong with your preps. Check the troubleshoot section into the manufacturer manual of your plasmid prep kit for similar weird situations. 

-Ensure bacterial cultures are properly inclined and the shaking is constant at > 200 RPM (ideal 220-230) for maximize bugs growth. 

I can finally add few alternative technical suggestions. You can setup 2-3 or even more preps for the same clone and you can perform the preps separately and join preps together finally.

Indeed, if you're performing a midi-maxi prep you'll have a pellet after the isopropylic alcohol precipitation and ethanol washes, you can cut a p1000 tips with enough width to ensure you can aspirate the whole pellet and then you can aspirate all the pellets and place all of them into a single 2mL vial. You can then centrifuge again and air dry the "megapellet" and resuspend it into desired volume of nuclease free water.

if you're performing minipreps with an in-colums final DNA eluition step, you can place all the eluition products of multiple preps into a single 2 mL vial and precipitate the plasmids by adding 1/10 sodium acetate 3.5 M pH 5.4 (also 3.0 M ph 5.5 works fine) and 2 volumes of absolute ethanol. Wash the pellet with 70% ethanol, air dry the pellet and again you can resuspend into the proper volume of nuclease free water.

If you want also to rise your plasmid purity you can also perform a phenol-chloroform extraction instead of Na-Ac/ethanol precipitation. In this way you'll have a increased amount of plasmid into desired volume of solution.

If you are using a low copy plasmid you can use a chloramphenicol protocol:

- mini culture on night

- maxi culture on the day

- on the evening you add your antibio, fresh medium and chloramphenicol: the bacteria growth will be blocked but not the synthesis of you're plasmid. it's working very well

thanks alot. this discussion helped alot thanks to Laurent Vanhille Paolo Scarpelli and Yeu Yau

Dear Qari Muhaamd Imran, Can you please tell the name of your plasmid. Some plasmids are of very low copy number, You can culture it for longer time and get more number of colonies, Secondly you can take big amount of culture to make pallet. Sometimes 2 or 3 ml is not enough and need to take nearly 8-10 mL. I hope it will help you.