Plasmid extraction from E.coli culture - (Jun/17/2004 )

Hi all,

I have to extract and purify a 5.5 kb plasmid from an E. coli culture. But I have some questions:

1. What is the best choice to spread an agar plate from an E. coli glycerol stock (-80 ºC)?

2. Other times I thawed the stock and spread 2-5 uL onto the agar plate. Could thaw and refreeze several times the same stock damage the plasmid for subsequent use in yeast transfection?

3. Why the plasmid concentration was fine with the first extractions and very poor after have thawed and refreeze several times the glycerol stocks?

Thanks a lot for all suggestions

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Hi celvas,

I guess the plasmid is 5.5kb and not kDa (kDa is for proteins)

First off, NEVER ALLOW YOUR GLYCEROLS TO THAW. Take the tube out of the -80'C, put it straight on ice and use the tip of the pippete to transfer as small amount (should be solid, ie very cold) into fresh medium (in case of liquid culture) or use your loop to streak it on an agar plate. Again, NEVER THAW THE GLYCEROL STOCKS, they should be out and back in the -80'C in 2-3mins MAX!

Regarding streaking on a plate or straight into a liquid culture, I would certainly go for an agarose plate first and then the next day pick a colony into a fresh 3ml culture (and you know the rest).

Regarding the plasmid concentration after the miniprep bear in mind a few things:
a. DO NOT THAW GLYCEROLS (again), cell viability decreases considerably and in come cases the plasmid itself might be lost
b. Having said that, since you are getting growth in your liquid cultures, there shouldn't be a question of the plasmid maintained in the cells (provided ofcourse you used the correct antibiotic)
c. Sometimes by increasing the concentration of the antibiotic you might get better yields (or slightly better expression if you are doing protein purification work). The reason is unclear (to me, at least) but it is believed that the more antibiotic, the better plasmids are maintained and the stronger the antibiotic gene and/or cloned gene are expressed.
d. Keep off the vortex during the miniprep (if you do it manually), mix everything by gentle invertion of the tube. This is very important!!!

This is all I can thing of at the moment. Good luck.

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Lot of thanks for your kind reply. It helps me for good practical techniques. Let me ask you an other question:
As the plasmid produced by thawed-refreeze glycerol stock has been sequenced, I need to work with it. The question is:

If I streak an agar plate containing selective antibiotic and the bacteria growth, can I think that these bacteria are suitable for quality plamid extraction? Or do you recommend me for sequencing an other clone stock?

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hi

you don't have to use agarose to make a plate, agar is the one people usually use, unless you have special purpose.

bacteria bearing a plasmid grow do not mean they are good for high quality plasmid isolation - of course it depends on what purpose you use it for - sequencing would need a higher quality than general cloning. sometimes it also happens that one bacteria is good for plasmid A but not good for plasmid B, plamsid size, or copy number also play a role. different host (bacteria) have different genotype which would affect you plasmid "quality" and yield. for example some bacteria have wt methylation gene, say dam gene for methylating adenine of GATC, no matter how pure your plasmid DNA is, you can't cut it using restriction enzyme like BclI which recognizes TGATCA, b/c BclI is sensitive to this methylation. Having you plasmid DNA methylated is not always bad, sometimes people need methylated plasmid to transform a special host, other the unmethylated plasmid would be restricted.

the newer E. coli host like DH5alpha, DH10B, XL1-Blue... have many wt genes inactivated which make your plasmid DNA easier to be purified, and usually fit most, if not all, of your work.

hope this helps,

Mike

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Would like to voice up my enquiries: Ive did plasmid extraction for the 1st time on a gram positive bacteria. Ive gotten results, clear bands but I do not know how to interprete the results. What should I do? Ive referred to my supervisor but she asked me to ask my freinds. According to my friends, they said make comparisons about the size and see whether there's any resemblance in any of the isolates. What else can be done?


By the way, are there any methods to optimize the extraction? I used TAE 1X instead of TBE.


Thank you, hope to hear some feebacks from you all.

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Hi,

I have just signed up as member and I find that this forum is indeed very useful. I learn more of certain things which I've knew. Would like to post few questions to INVISIBLE SURFER regarding your reply to celvas.

"Regarding streaking on a plate or straight into a liquid culture, I would certainly go for an agarose plate first and then the next day pick a colony into a fresh 3ml culture (and you know the rest)".

Was the plate and culture you'r mentioning here is LB? Should I supplement them with ampicilin (concentration)? Should I add IPTG/X-Gal as well on the plate?

Thank you. Really hope to hear some feedbacks from you soon......

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this is for rachel - whether you use IPTG or X-gal depends on the plasmid and the cells you're using. Use X-gal if you are using a plasmid that can do LacZ blue/white colour selection, most commercial plasmids have this feature, eg pUC18/19, pCR2.1, pBluescript or any of their derivatives. IPTG is also involved with the LacZ system, its a lactose analogue. You only need to add it for some cell types, eg Top10F` require IPTG, Top10 cells don't.
Whether you add ampicillin also depends on the plasmid, and what antibiotic resistances it has. Look at the plasmid map and you will be able to see if it has LacZ and amp resistance.
for jazzlyn - I haven't heard of using TBE or TAE for plasmid extraction - do you mean TE buffer? If you use a kit, then just follow the directions. If you use the plasmid alkaline miniprep method (solutions 1, 2 and 3) then post your method so we can see. What is the name of the plasmid you have isolated? To know if your bands on the gel mean anything, you have to know the size of your plasmid. Is there a gene inserted into your plasmid, or is it uncut?

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Thanks scrat...

will check more on those info you've given me.

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