Transformation doesn't work (no insert where it should be...)
 

Hy =)

So, we have a plasmid with an insert. (the insert is allready in the plasmid, this plasmid is from a maxiprep long long time ago. so we need now more of this plasmid).

i made a transformation, first in JM109 then in Top10 Comp. Bacterias. (25ng Plasmid, 20minutes on ice, heat shock, 1h / 37°C / 200rpm like usually).
then i gave the bacterias on LB-Agar plates with ampicillin (100ug/ml final conc.).
Then i put the plates in a 37°C inkubator over night.
so, my colonies are reeeeally small (usually i get bigger colonies). but, i make a miniprep, then a restriction enzyme digestion (EcoR1) for 2h / 37°C and make a gel.
the original plasmid i digested also and put it on the gel too.

the original plasmid gave me two bands like expected, but the minipreps give me only one band (for the plasmid), they hadn't a band for the insert....
(but the insert should be in the plasmid, so no ligation faults (because of the controll of the original plasmid).

Can someone help me? what can i change? why i can't find the insert in the plasmid? is there a problem, that my colonies are so small? what can i change, that i get bigger colonies? (i used LB-Agar with NaCl, tryptone, Yeast Extract and Agar)

thanks

Re: Transformation doesn't work (no insert where it should be...)
 

so, i found out what the problem was;-) the LB-Media.... the Tryptone and the Yeast Extract are 20 years old... thats too old^^ with new tryptone and yeast extract it works perfect^^

Re: Transformation doesn't work (no insert where it should be...)
 

20 years old! OMFG! xDDD

Re: Transformation doesn't work (no insert where it should be...)
 

in our lab, EVERYTHING is old!! old, older, our chemicals/reagents!! -.- (and if something doesn't work, one of the lab technicians is guilty, not the old things^^)

Re: Transformation doesn't work (no insert where it should be...)
 

You need do it again,it isn't problem

Re: Transformation doesn't work (no insert where it should be...)
 

Did you guys like, actually have Luria making your media for you? LOL?