Protocol Online logo
Top : Forum Archives: : Molecular Biology

Re-innoculate overnight culture for miniprep - (Feb/19/2006 )

I got my clone and I have confirmed it with the restriction digestion now want to determine its reading frame for sequencing. But my plasmid DNA is almost finished and all consumed in restriction digestion, DNA concentration determination and Sequencing Reaction.
But I have plasmid L.B culture about 1,5 ml from where i got my plasmid DNA sequencing and other Test. But i have kept it in cold room at 4 C. I want to ask that can I used 100 microlit of this culture to inoculate new LB media for fresh culture to get plasmid for transformation into BL21 culture cells for getting expression. Or I have to make again a new tranforamation for getting fresh culture.
Whats would be negai┬Ętive effect of using 5 to 6 days old culture in cold room to incolulte fresh LB meida to get plasmid for expression transformation.
How long LB agar plates containing colned clones can be kept and used to isolate cloned plamid.
best regards

-anu1-

It is perfect fine to innoculate the bacteria. This is exactly what people do for maxiprep after miniprep.

Colonies on plates can survive from a few weeks to months.

-pcrman-

QUOTE (pcrman @ Feb 19 2006, 12:27 PM)
It is perfect fine to innoculate the bacteria. This is exactly what people do for maxiprep after miniprep.

Colonies on plates can survive from a few weeks to months.


I just got four colonies of recombinant plasmid and i took whole colony for culture and I have no plate to again reinoculate. But I have glyserol culture at - 70 and culture with out glyserol frozen at -20, What i do now. If I make a plellet of culture stored in cold room to again spread on plate and take colony from that agar palte to get pure plasmid for transformation.
will it be ok or any other way please suggest me.
best regards

-anu1-

QUOTE
I want to ask that can I used 100 microlit of this culture to inoculate new LB media for fresh culture to get plasmid for transformation into BL21 culture cells for getting expression.

I meant what you said above. This is the easiest way.

-pcrman-

how about taking the final amount of your plasmid and re-transorming your fresh compotent cells? This is an option if your cells are not ok.

-ML1975-