Added E coli to plasmid by mistake - How to get plasmid back? (Aug/08/2006 )
I received plasmids from another lab on filter paper. And I have put the filter paper in a tube and dissolved the plasmids in TE in a total volume of 30ul. Due to my careless manipulation, I added 25 ul competent E coli into the tube containing my plasmids. The plasmids are important for me.
Does any one know if the the solution will be suitable to transform E.coli or how to rescue my plasmids from the solution?
Thanks,
If you want to separate DNA from bacteria, I would just spin the mix down at 13000 rpm for 5 to 10 minutes. teh plasmid should stay in solution and the bacteria would pellet down.
QUICK! Add some SOC and plate something out! 
:-)
:-)swanny,
Are there big differences between SOC and LB in the initial culturing during transformation?
I have tried to transform cells using LB as initial culturing medium but plated out nothing
Thanks,
QUICK! Add some SOC and plate something out!
:-)swanny,
Are there big differences between SOC and LB in the initial culturing during transformation?
I have tried to transform cells using LB as initial culturing medium but plated out nothing
Thanks,
There sure is! Sorry to be the bearer of bad news, but your lack of success could very well be down to using LB rather than SOC.
I don't know exactly *why* (I'm sure someone here knows!), but I think it's linked to the amount of salt (LB=1%, SOC=0.05%). Remember, your cells have just been through some fairly harsh times (making competent, snap-freezing, heat-shock), and they are just getting back into a nice rich medium. They are still fragile (weak membrane? / pores in membrane?), so you need to be gentle to them. By the time they've had the initial recovery step, they should be robust enough to survive the effects of the antibiotic on your plate.
I agree with canalon if u need quick and accurate result u have u do like that,because eventhough u add LB or other medium is notsufficient for transforming.tell me one thing have u done this inLAFU chamber ?(ecoli cells to plasmid)
you could heatshock what you've got!!!!
Add colour 2 ur life,
I have some difficulties understanding English, so would you please show me your methods in a more detailed way. And what is a LAFU chamber? And I have added 25 ul competent cells into my plasmids by accident in a 1.5ml EP tube.
Thanks,