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Преобразование плазмидной ДНК в E.coli, с использованием метода теплового шока

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Department of Physiology and Biophysics, University of California, Irvine (UCI)

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Cite this Article: Преобразование плазмидной ДНК в E.coli, с использованием метода теплового шока

Froger, A., Hall, J. E. Transformation of Plasmid DNA into E. coli Using the Heat Shock Method. J. Vis. Exp. (6), e253, doi:10.3791/253 (2007).

Abstract: Преобразование плазмидной ДНК в E.coli, с использованием метода теплового шока

Преобразование плазмиды ДНК в E.coli, методом теплового шока основную технику молекулярной биологии. Она состоит из вставки иностранных плазмиды или перевязки продукта на бактерии. Это видео протокол описывает традиционный метод преобразования с использованием коммерчески доступных химически компетентных бактерий из Genlantis. После короткой инкубации в лед, смеси химически компетентных бактерии и ДНК находится на 42 ° С в течение 45 секунд (тепловой шок), а затем помещен обратно в лед. SOC СМИ добавляется и трансформированные клетки инкубируют при 37 ° С в течение 30 мин при перемешивании. Чтобы быть уверенным в изоляции колоний, независимо от эффективности трансформации, две величины трансформированных бактерий покрытием. Этот традиционный протокол может быть успешно использован для преобразования самых коммерчески доступных компетентных бактерий. Turbocells от Genlantis также может быть использован в романе 3-х минутах преобразование протоколов, описанных в инструкции по эксплуатации.

Disclosures: Преобразование плазмидной ДНК в E.coli, с использованием метода теплового шока

Ask the Author: Преобразование плазмидной ДНК в E.coli, с использованием метода теплового шока

9 Comments

I have never seen or heard anyone use beads to create a spread plate. Is that a new technique that has not reached my university yet, or is it an outdated technique that scientists don't use anymore?

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Posted by: AnonymousFebruary 1, 2008, 7:57 PM

hi my neam isalirezababazade . iam student in unversity tehran . what do you teransfer factors F(negativ)in the e.coli?why transfe geneboth factor?

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Posted by: aliDecember 24, 2008, 9:41 AM

People use it all the time. Ask in the labs around, I am sure you will find people who use it.

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Posted by: AnonymousFebruary 1, 2008, 8:11 PM

I haven't seen it either so you're not alone.

I'm wondering if it isn't easily to use a spreader in terms of cleanup and reducing contamination? 

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Posted by: AnonymousMarch 9, 2008, 8:45 PM

Copacabana method for spreading E. coli and yeast colonies
Mark T. Worthington, Roger Qi Luo, and Jared Pelo
BioTechniques Vol. 30, No. 4: pp 738-741 (Apr 2001)

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Posted by: ozgur g.September 9, 2008, 9:04 PM

chemical dna transformation and plasmid curing of E.coli

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Posted by: ali sabahJanuary 11, 2012, 11:49 PM

why don't the flame is open? Don't you consider the contamination risk, or do you think that it is not necessary??

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Posted by: zerrinSeptember 9, 2008, 4:22 PM

Please look more carefully. The bunsen burner is clearly on and the blue flame is clearly visible in the video. It is on at all stages which are succeptible to contamination.   

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Posted by: labSeptember 16, 2008, 8:57 PM

That was indeed a great thing for the beginners in this field. Good job!

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Posted by: kamal prajapatiOctober 22, 2008, 8:11 PM

double stranded plasmid can be fntered into host

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Posted by: khemJune 10, 2010, 6:00 AM

Beads are used as well as scoops, i prefare scoops because i can be sure that everything is spread nice and equal. some technician use beads, you need to do the right motion to make it work :D

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Posted by: MuadJanuary 25, 2012, 3:59 AM

Why we are keep the mixture of competent cells and plasmid DNA at 42c for 45 minutes?

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Posted by: NagarjunaFebruary 22, 2012, 8:56 AM

45 seconds not minutes XD
Useful, more protocols should be explain like this one
thaks

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Posted by: AdrianApril 12, 2012, 7:48 PM

Where is gloves to prevent contamination?????

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Posted by: OHIONET O.May 10, 2012, 4:35 PM

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