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Asking help for purifying plasmids - (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 solution will be suitable to transform E.coli or how to purify my plasmids from the solution?
Thanks,

-coata-

I wouldn't try to purify them. Keep the tube and transform (chemically) new cells with 1 ul of the cells + TE + plasmid mix. I'll bet it will work if you have good competent cells.

I should add that the plasmid is unlikely to be very stable in this mixture. You should do this promptly, or else freeze the sample at -80 until you have some competent cells available.

-phage434-

QUOTE (phage434 @ Aug 8 2006, 07:45 AM)
I wouldn't try to purify them. Keep the tube and transform (chemically) new cells with 1 ul of the cells + TE + plasmid mix. I'll bet it will work if you have good competent cells.

I should add that the plasmid is unlikely to be very stable in this mixture. You should do this promptly, or else freeze the sample at -80 until you have some competent cells available.

phage434,
I have tried to transform commercial competent cells.
And I have tried different proportions of the mixture. Those are 2.5ul plasmid mixture with 25ul cells, 5 ul plasmid mixture with 25ul cells and 7.5 ul plasmid mixture with 50ul cells. The competent cells I used were purchased from a commercial company. My transformation protocol is
1 add the DNA into competent cells
2 keep the mixture on ice for 30 min
3 heat shock in 42 centigrade for 90s
4 quickly transfer the mixture into ice and cool for 2.5 min
5 add 500ul LB without antibiotics and incubate in 37 centigrade for 45 min
6 plate 100ul of the mixture on plates with antibiotics added and incubate in incubator under 37 centigrade overnight
After the procedures, I didn't see a clone. So it may be troubling,
I have kept the mixture of plasmids in -70 refrigerator.
Have you done that before? Are there any special cautions should taken into account?
Thanks

-coata-

QUOTE (phage434 @ Aug 8 2006, 07:45 AM)
I wouldn't try to purify them. Keep the tube and transform (chemically) new cells with 1 ul of the cells + TE + plasmid mix. I'll bet it will work if you have good competent cells.

I should add that the plasmid is unlikely to be very stable in this mixture. You should do this promptly, or else freeze the sample at -80 until you have some competent cells available.

In addition, will the concentration of plasmids in such a mixture be too low for transformation?
It is the first time for me to harvest plasmids from a filter paper, so thanks for your patience and kindly help,

-coata-

I'd say you are using too much of your mixture for transformation. Try 1 ul or less in 25 ul of cells. Also, a 90 second heat shock sounds unusually long. Try 45-60 seconds.
Are you sure you are using the correct antibiotic? If anything other than ampicillin, I would grow the cells without selection for longer than 45 minutes -- try 1.5 or 2 hours.
Also, I would use SOC not LB for initial culturing, and probably a smaller volume, which will make it easier to plate the entire amount. I use 250 ul of SOC with 25 ul of cells.

-phage434-

QUOTE (phage434 @ Aug 8 2006, 07:53 PM)
I'd say you are using too much of your mixture for transformation. Try 1 ul or less in 25 ul of cells. Also, a 90 second heat shock sounds unusually long. Try 45-60 seconds.
Are you sure you are using the correct antibiotic? If anything other than ampicillin, I would grow the cells without selection for longer than 45 minutes -- try 1.5 or 2 hours.
Also, I would use SOC not LB for initial culturing, and probably a smaller volume, which will make it easier to plate the entire amount. I use 250 ul of SOC with 25 ul of cells.

phage434,
Do you mean that I should use SOC without antibiotics for initial culturing and LB plates with antibiotics for following overnight plating?
The antibiotic I'm using is kanamycin. The concentration is 30ug/ul
And I saved the initial culture I got the day before yesterday under 4 centigrade and plated them yesterday. This time I used a larger volume of initial culture, that is 300ul initial culture instead of 100ul. But I still cannot see a clone after overnight plating.

-coata-

Yes, that was what I meant. For Kan, I would definitely grow longer than 45 minutes without antibiotic. Since you don't care about selecting individual clones, overgrowing the initial culture slightly will just give you more chance to plate out resistant colonies.
I trust you are certain that the plasmids you are attempting to recover are, in fact, kan resistant.

-phage434-

QUOTE (phage434 @ Aug 9 2006, 12:05 PM)
Yes, that was what I meant. For Kan, I would definitely grow longer than 45 minutes without antibiotic. Since you don't care about selecting individual clones, overgrowing the initial culture slightly will just give you more chance to plate out resistant colonies.
I trust you are certain that the plasmids you are attempting to recover are, in fact, kan resistant.

phage434,
Where is your competent cells come, prepared by yourself or purchased from company?

-coasta-

We use Invitrogen Top10 chemically competent cells, or make them as here:

http://openwetware.org/wiki/TOP10_chemically_competent_cells

-phage434-

QUOTE (phage434 @ Aug 9 2006, 09:37 PM)
We use Invitrogen Top10 chemically competent cells, or make them as here:

http://openwetware.org/wiki/TOP10_chemically_competent_cells

phage434,
It is disappointed for me. Though I transformed DH5a competent cells in accordance to your protocol yesterday, I didn't get a single clone this morning.
So are there any further cautions added for transformation?

-coasta-