Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Cloning

HELP!Mini preps resulting in low yields where previously high. - (Nov/08/2010 )

Pages: 1 2 Next

Hi,

I'm having serious issues with mini preps. Suddenly each plasmid Iíve tried to grow has a concentration of 200ng/ul or less (they used to be well above 1000ng/ul.)

I should note, the agar plate colonies look ok. However the mini prep broths are always silty looking and the pellets re-suspend in chimney like formations and I can tell before I run the minis that the concentration will be low.


Iíve tried the following,

Plasmids with different antibiotic resistance Kan and Amp.
Running each with different antibiotic concentrations and at different time points from 10hrs-24hrs.
Different types of LB-broth 10,5,5 and 10,5,10
Different competent cells, in-house, dH5alphas, Stbl3
Different ratios of LB-broth to bacteria
Proper washing of bottles to ensure no trace of detergents
I use Invitrogen mini-prep kits (tried more than one batch number)
I've grown the broth alone and inoculated LB broth with agar form a plate to rule out some low level contaminant.


Any advice would be great!

Lou

-louiseg150-

Do your plasmids express any genes?

-perneseblue-

Hi,

Please transform bacteria on new fresh plate and go for mini prep, Your problem will be solved.

Regards,

Luciana

-mole-

I have not experienced such a problem. But, the only thing comes to my mind is the shaker. It looks you did not do anything about it. The shaker may not shake fast enough or the temperature within it may not be accurate or stable. Give a try for a different one. I hope it helps

-GeneTurk-

Phage434 suggests that this might be phage contamination. Go Phage!

-phage434-

phage434 on Tue Nov 9 23:11:34 2010 said:


Phage434 suggests that this might be phage contamination. Go Phage!


In response, UV irradiation of all culturing glassware should exorcise this infestation.

Begone phage, Begone! The energy of UV light compels you. :lol:

-perneseblue-

perneseblue on Wed Nov 10 04:44:01 2010 said:


In response, UV irradiation of all culturing glassware should exorcise this infestation.

Begone phage, Begone! The energy of UV light compels you. :lol:


Hmn... this sounds like a phage exorcism voodoo mantra... phage might be gone but then we still need our phage434 here. ^_^

But then, seriously, is phage contamination frequently happen? How do we know when is phage contamination and how we can prove it? This sounds scary...

-adrian kohsf-

Could be that you have detergent residues on the glassware. Was the glassware thoroughly and rinse several times in distilled water (some say to autoclave in distilled H2O).

-bob1-

adrian kohsf on Wed Nov 10 16:59:56 2010 said:


But then, seriously, is phage contamination frequently happen? How do we know when is phage contamination and how we can prove it? This sounds scary...


Phage infestation are rare (unless somebody in the lab/corridor actually working with phage).The symptoms of an infestation is typically goes something like "the culture was growing fine, when it just stopped growing and started being less cloudy/going clear.."

Usually culture failure are caused by
-leaky expression of some toxic gene.
-glassware contaminated by detergent.
-contamination by competitive/predatorial bacteria.
-phage infestation.

-perneseblue-

perneseblue on Thu Nov 11 04:24:04 2010 said:


adrian kohsf on Wed Nov 10 16:59:56 2010 said:


But then, seriously, is phage contamination frequently happen? How do we know when is phage contamination and how we can prove it? This sounds scary...


Phage infestation are rare (unless somebody in the lab/corridor actually working with phage).The symptoms of an infestation is typically goes something like "the culture was growing fine, when it just stopped growing and started being less cloudy/going clear.."

Usually culture failure are caused by
-leaky expression of some toxic gene.
-glassware contaminated by detergent.
-contamination by competitive/predatorial bacteria.
-phage infestation.


My newly purchased ROSETTA-GAMI 2(DE3)pLysS COMPETENT CELLS was growing fine, but it does relatively less cloudy when it grows on plate (compare with nova blue strains and BL21(DE3) strains. The transformation efficiency is also relatively low, and the culture revival success rate is also very low. Do you think is possible of phage infestation?

Thanks.

-adrian kohsf-
Pages: 1 2 Next