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Qiagen midi prepare low yield - (Dec/16/2009 )

I met a trouble on DNA midi or max prepare at this point.

That I use Qiagen high speed midi or max DNA prepare Kit to my Pbluescript based vector. But I got very low yield. close to 10-20ug/150mL bac after culture 14-16hours. The miniprepare is good as normal before I start midi or max culture them. I had this vector with several different inserts. I can get some of them work. but most of them not.

by the way, I also had another Kan+ resistant vector CFP-C1 to purified at the same time. It worked well. So I do not think problem from the reagents or protocols.

Please give me a clue. Thanks

-newboy-

As far as i know pBluescript are Amp-based vectors? ...aren't they?

Amp-resistance sometimes makes problems since the beta-lactamase is secreted into the broth and therefore the selection gets weaker and weaker. What concentration of antibiotics you are using?

But, since you mentioned that mini-prep works quite well it might be that you have problems lysing your cells ...this can be often a problem with e.g. JM-strains. Lysis is a very critical step and often more difficult in larger scales. Are you using the Qiagen kits with the LyseBlue-Solution?

Maybe you can give more details about your procedures? ...help willl be much easier.

Regards,
p

-pDNA-

I've got this problem sometimes and I agree with pDNA. However, the best way to solve that is re-transform your plasmid to fresh competent cells and you can either spread them on the selection plate or grow directly the transformed cells in the large volume medium with Amp, then miniprep.

-Quasimondo-

Thanks for those suggestions. I try re-transformation. It works sometimes. BTW, the Amp conc I used is 100ug/mL

I can get my mini prepare well at any time. I tried to make mini prepare through 4-5mL of the max cultured bac. I can see very less plasmid. I guess it was Amp or bac problem. I wander if I have the problems at here.

I pick out the clones, mini culture in 4-5 ml with Amp 100ug/ml for 10-12 hours, then save 500uL in 4oC, others made mini prepare, enzyme check the plasmid correct or not with gel elec. then I take out the 500uL bac from 4oC (usually 5-10 hours later), apply 100-200uL into 100-200mL LB for midi or max culture. then I cannot get plasmid here. I doubt if I should keep them in 4oC. or I need culture them in 37oC continuously!?

-newboy-

pDNA on Dec 27 2009, 01:09 PM said:

As far as i know pBluescript are Amp-based vectors? ...aren't they?

Amp-resistance sometimes makes problems since the beta-lactamase is secreted into the broth and therefore the selection gets weaker and weaker. What concentration of antibiotics you are using?

But, since you mentioned that mini-prep works quite well it might be that you have problems lysing your cells ...this can be often a problem with e.g. JM-strains. Lysis is a very critical step and often more difficult in larger scales. Are you using the Qiagen kits with the LyseBlue-Solution?

Maybe you can give more details about your procedures? ...help willl be much easier.

Regards,
p



thanks for your reply, yes they are Amp based, I use 100ug/mL for selection and culture

I guess lysis may be fine for mine because all my Kan resis plasmids work well with Qiagen mid prepare kit. Yes the hispeed kit with lyse blue with it in P1

-newboy-

newboy on Dec 29 2009, 04:35 PM said:

Thanks for those suggestions. I try re-transformation. It works sometimes. BTW, the Amp conc I used is 100ug/mL

I can get my mini prepare well at any time. I tried to make mini prepare through 4-5mL of the max cultured bac. I can see very less plasmid. I guess it was Amp or bac problem. I wander if I have the problems at here.

I pick out the clones, mini culture in 4-5 ml with Amp 100ug/ml for 10-12 hours, then save 500uL in 4oC, others made mini prepare, enzyme check the plasmid correct or not with gel elec. then I take out the 500uL bac from 4oC (usually 5-10 hours later), apply 100-200uL into 100-200mL LB for midi or max culture. then I cannot get plasmid here. I doubt if I should keep them in 4oC. or I need culture them in 37oC continuously!?


i would not do it that way ...during the storage at 4C you might lose your plasmid ...anerobic conditions will promote plasmid loss!

I would make real glycerin stocks or masterplates instead of the procedure you describe!

Regards,
p

-pDNA-

Thanks

I tried to freeze bac down to -80oC. then mini culture in 5mL. then apply 200uL to 200mL to perform max culture. I can get some plasmid after this. but still not good enough. I can get 50-100 ug plasmid with midi kit.

by the way, what is the masterplates?


newboy on Dec 29 2009, 10:39 AM said:

pDNA on Dec 27 2009, 01:09 PM said:

As far as i know pBluescript are Amp-based vectors? ...aren't they?

Amp-resistance sometimes makes problems since the beta-lactamase is secreted into the broth and therefore the selection gets weaker and weaker. What concentration of antibiotics you are using?

But, since you mentioned that mini-prep works quite well it might be that you have problems lysing your cells ...this can be often a problem with e.g. JM-strains. Lysis is a very critical step and often more difficult in larger scales. Are you using the Qiagen kits with the LyseBlue-Solution?

Maybe you can give more details about your procedures? ...help willl be much easier.

Regards,
p



thanks for your reply, yes they are Amp based, I use 100ug/mL for selection and culture

I guess lysis may be fine for mine because all my Kan resis plasmids work well with Qiagen mid prepare kit. Yes the hispeed kit with lyse blue with it in P1

-newboy-