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To what extent competent cells be thawed before transformation?


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4 replies to this topic

#1 SF_HK

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Posted 03 March 2009 - 06:18 PM

Hi,

Recently I have not been getting colonbies on my plate, tried out new ligase, new ligation buffer, different vector to insert ratio etc but just 3-6 empty colonies per plate. Is there a way to thaw frozen competent cells? I usually haw them on ice and when they are completely thaw, I add the ligaion mixture. I heard some people add teh ligation mixture when hey are semi-thawed?

#2 scolix

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Posted 06 March 2009 - 11:04 AM

I thaw cells on ice for 10-15 min. depending if I have time, or simply add the ligation mixture when it is semi thawed, its the same thing. The idea is to mix cells and ligation mixture.

#3 molgen

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Posted 08 March 2009 - 04:51 AM

I have a stupid question for you:
Is the selective gene the same as the antibiotic?

I once tried growing Kanamycin resistant on an ampicillin plate (with out much success).
I learned a good lesson from it never take for granted what people tell you, always check it your self!

Edited by molgen, 08 March 2009 - 04:51 AM.


#4 HomeBrew

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Posted 08 March 2009 - 06:03 AM

Is the selective gene the same as the antibiotic?


No. The "selective gene" is a gene that confers resistance or sensitivity to some substance, not always an antibiotic (the "selective substance", if you like). There are genes that confer resistance to substances that aren't antibiotics, like HgCl2, and there are genes that confer sensitivity to other substances, like sucrose, that allows one to counter select.

#5 Nrelo

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Posted 08 March 2009 - 07:22 AM

I have a stupid question for you:
Is the selective gene the same as the antibiotic?

I once tried growing Kanamycin resistant on an ampicillin plate (with out much success).
I learned a good lesson from it never take for granted what people tell you, always check it your self!


i am continuing the work of my previous colleague, but her data already pissed me off becoz most of her works were not very reproducible. Good point by molgen.




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