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questions about transformation


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#1 KIT

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Posted 21 February 2011 - 12:26 AM

I have done an experinment on inserting DNA into pUC19, and put it into E.coli.
Afterwards, i grow the bacteria on LB plate with kanamycin. I then pick the colonies and grow it in LB medium with kanamycin
But then those bacteria cannot grow in the medium, i wanna ask why?

Some of the medium with bacterial grwoth, i then isolate the plasmid using plasmid purification kit with silica-gel membrane. Why the yield is not high?

Thanks so much

#2 protolder

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Posted 21 February 2011 - 12:50 AM

Hola, I was not sure but I have checked it, and pUC19 has ampicilin resistance. Or are you clonning any hydrolitic enzyme for Kanamycin?. Buen día

#3 KIT

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Posted 21 February 2011 - 02:23 AM

yes, the insert contain genes that code for kanamycin anti-resistance

#4 protolder

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Posted 21 February 2011 - 03:34 AM

yes, the insert contain genes that code for kanamycin anti-resistance

Hola, but if you have cloned kana genes under the promoter, you have to induce it with IPTG because this expression could be repressed and resistence coulnīt be showed. To get more plasmid have to grow the culture untill 2-3 OD unts at 600nm. Buena suerte

#5 Qundo12

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Posted 07 March 2011 - 01:02 AM


yes, the insert contain genes that code for kanamycin anti-resistance

Hola, but if you have cloned kana genes under the promoter, you have to induce it with IPTG because this expression could be repressed and resistence coulnīt be showed. To get more plasmid have to grow the culture untill 2-3 OD unts at 600nm. Buena suerte

He's right about the promoter. Normally you could digest out the Kanamycin resistance gene from other plasmid with its own promoter, and this one is constitutive. If you consider that, there are two possibilities:
1. The donor plasmid which contains the KanR was carried over after the gel extraction, if it's the low copy number plasmid, this could explain for the case that you get the low yield of pladmid. It happened to me many times, even though I excise out the low band, some of the intact plasmid still contaminated.
2. You've got the contamination, they can resist to the Kan on the plate but they will die if you grow them in the medium.

I suggest to spread your transformed cells on the Kan + Amp plate for the accurate screening.
If then you still have the low yield of plasmid, you can do the control experiment by prep the original pUC, it's the high copy number plasmid. In the case you get the low yield, you should check your kit, it might be in trouble.
Some point mutations can reduce the copy number so you get the low yield, but if it happens every time, mutation can't be the reason.




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