I transformed a plasmid DNA into my own made competent cells (DH5a) and i got no colonies neither in my sample plate nor in positive control.
I prepared competent cells using following protocol :
I picked a single colony from DH5a and inoculated 10mL LB with it and it was incubated on 37C at 120rpm overnight.
Then i refreshed it by inoculating 10 mL LB with 100 uL of overnight incubated liquid and it is shaken at 120rpm and 37C to reach OD= 0.4 - 0.6. Then i poured the liquid into 1.5 mL tubes and centrifuged them at 6000rpm for 2 mins. then i resuspend the pelletes first in 1 mL CaCl2 100mM and put it on ice for 30 mins then centrifuged it and discard the sup then in 700 ul and then in 300 ul.After adding 300 ul i added 75ul glycerol and stored it in -20.
I would be appreciated if anyone helps.
Hi, 6000 rpm is good when you want to isolate plasmids from bacteria, but if you want to keep them alive you shouldn't use the speed more then 4000 rpm, better to stick to 3000-3500 rpm. You probably killed your competent cell, considering that you weakening their cell walls in order to make them compitent.
Hi, I have used another protocol where I wash cells thrice with MgCl2.CaCl2 (80mM, 20mM) and then finally re-suspend the pellet in 0.1M CaCl2.
Apart from this long storage of competent cells in -20 might too effect their competency, rather if possible use -80. Hope this helps.
Timur Yergaliyev Thanks for your help
Shubhashish Chakraborty would you mind explain your protocol with more details please?
So the protocol is adapted from Sambrook and Russel book. Its just that they refereed single wash whereas we carry out 3 wash with MgCl2.CaCl2. For further details you can check out the book.
Hi, When you cultured bacteria in plate ,the concentration of antibiotics was apropriate ?
It is very important when making chemically competent cells that the cells be kept ice cold. Be sure your centrifuge is chilled, your tubes are ice cold and the solutions are all cold.
There are numerous variations on the CaCl2 procedure, in general they all work fine some work slightly better for some strains than for others. You can store competent cells at 4oC for 1-2 days before using. For longer term storage you need to have added some glycerol or DMSO to the final resuspension buffer and store at -80C. But that is only for storage and not necessary if you intend to use them quickly. Storing at -20C is generally not a good idea, but might be fine for a few days.
Thanks to all
Finally, i got colonies.
I think the problem was storing the cells at -20, because when i made competent cells and transformed DNA in the same day it worked.
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