3 replies to this topic

[Wonyong]

Hi guys


I am trying to clone a gene of my interest into retroviral vector.

While maxipreping vector by transforming DH5a with vector DNA stock, I found strange debris in my liquid culture.

It looks like fibers floating in the culture, and when I centrifuge it aggregates with my bacterial pellet and don't resolve back.

I first thought it might be contamination and PCIAA + EtOH ppted the stock DNA. But the issue remains.

The vector is about 6.5kb long and has ampicillin resistant gene for selection.

Neither media or amp seem wrong since they worked perfectly in other cultures. I also maxipreped another clone which uses same vector but with a cloned DNA in it with no problem.


Thanks for reading and please get me some idea. I really need this cloned.

Edited by Wonyong, 26 March 2009 - 06:07 AM.

[bob1]

It could be fibers from cotton-wool used to plug the necks of flasks when growing the cells.  

If it isn't a contamination that is living, and it doesn't dissolve, then is there an issue?   It probably won't affect your end result.

[Wonyong]

Sorry that my first description might have been too simple.

They look like fibers but they grow together with bacteria. They were never there at the beginning of the culture.

They severely affect the productivity of my experiment since I cannot get any desired vectors from the maxiprep.

So that they seem to grow, it might be contamination but I cannot rule out it might be the debris from the death of bacteria.

Or is it possible the bacterial phenotype changes in suboptimal ampicillin level?

[lab_member]

Just want to ask a stupid question: did you use your liquid culture immediately out of the incubator or did you put on you bench or store somewhere for a few days? Because last time when I stored my bacteria too long, it died, though I think i didn't do any prep, but i think dead bacteria gives low yield.

Could it be the suboptimal concentration of ampicillin that caused your bacteria to overgrow (esp if you incubate too long like >=16 hrs), and when they reach a confluence too high, they will start to die and sometimes cell lysis would occur therefore affecting the yield...

Maybe it is really contamination like you said because of suboptimal ampicillin concentration, some other bacteria / fungi may grow and hinder the growth of your bacteria, resulting in low plasmid yield.

Another possibilty, which I think only has a very low possiblity, is that if you use soap / detergent to wash the flask before autoclaving and the rinse was not clean enough, the residual soap/detergent after autoclave would affect the bacteria. I have tried pouring some water into an autoclaved bottle in my lab and then bubbles formed rapidly, which I assume is residual soap. One of my lab colleagues also tried pouring LB into a flask and the bubbles formed were abnormal - we assume it was residual soap/detergent.

Hope it helps