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Transformation question


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#1 Ecoli0157

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Posted 19 February 2009 - 02:39 PM

Hi guys,

I need a library quality competent cells (~10^8CFUs/ug DNA) for my cloning purpose, but I have failed a couple of times and can't afford to keep buying commercial comp. cells.

Can I just use more comp cells (200ul/rxn) I have in my lab(manually preped) and set up more transformations to compensate for the low competency and look for the desired clone that way?

I did a test ligation where I re-ligated 60ng cut vector and transformed 12ng of this DNA. I got 300~400 colonies when spread without dilution on LB/amp. would this be a reasonable cell to use in terms of cell competency?

#2 pcrman

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Posted 19 February 2009 - 08:00 PM

300-400 colonies are pretty good yield. How many colonies do you want to screen? of course transforming more cells using more ligation reaction will increase the yield and representation of your library.

#3 T C

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Posted 19 February 2009 - 08:02 PM

Hey

Isn't 300-400 colonies good enough for your cloning purpose? More than this would give you a lawn and you won't be able to pick a single colony.

may I ask what cloning experiment is this?

TC

#4 WHR

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Posted 19 February 2009 - 09:55 PM

Hi guys,

I need a library quality competent cells (~10^8CFUs/ug DNA) for my cloning purpose,

I did a test ligation where I re-ligated 60ng cut vector and transformed 12ng of this DNA. I got 300~400 colonies when spread without dilution on LB/amp. would this be a reasonable cell to use in terms of cell competency?


Hi,
Your transformation efficiency is about 10^4-5, however, you need it to be 10^8. You may like to try using electroporation.

#5 T C

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Posted 19 February 2009 - 10:33 PM

Hmmm

Thats a good idea, but I use a different protocol to make electrocompetent cells. Will the normal chemical competent cells work?

TC

Hi,
Your transformation efficiency is about 10^4-5, however, you need it to be 10^8. You may like to try using electroporation.
[/quote]

#6 WHR

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Posted 19 February 2009 - 11:47 PM

Hmmm

Thats a good idea, but I use a different protocol to make electrocompetent cells. Will the normal chemical competent cells work?

TC


No, you can not use chemical competent cell for electroporation.

#7 Ecoli0157

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Posted 20 February 2009 - 07:59 AM

300-400 colonies are pretty good yield. How many colonies do you want to screen? of course transforming more cells using more ligation reaction will increase the yield and representation of your library.


I am not sure how I calculate that, but I have ~6MB genome which contains the desired insert of size 3 kb (I know this from S.blot). I am cutting it with EcoR1. EcoR1 does not cut within my insert.

Edited by Ecoli0157, 20 February 2009 - 08:07 AM.


#8 Ecoli0157

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Posted 20 February 2009 - 08:13 AM

Hey

Isn't 300-400 colonies good enough for your cloning purpose? More than this would give you a lawn and you won't be able to pick a single colony.

may I ask what cloning experiment is this?

TC


That would be good enough for a PCR cloning, but I am cloning the g.DNA, of which there is a very few fraction of my desired insert.

Well, 300~400 colonies were with the vector control DNA. I would expect a lot fewer colonies to come up from my ligations.
My experiment is to find a gene disrupted by a transposon and then sequence the flanking region. Then associate phenotype of that mutant with the identified gene.

#9 Ecoli0157

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Posted 20 February 2009 - 08:18 AM

Hi guys,

I need a library quality competent cells (~10^8CFUs/ug DNA) for my cloning purpose,

I did a test ligation where I re-ligated 60ng cut vector and transformed 12ng of this DNA. I got 300~400 colonies when spread without dilution on LB/amp. would this be a reasonable cell to use in terms of cell competency?


Hi,
Your transformation efficiency is about 10^4-5, however, you need it to be 10^8. You may like to try using electroporation.


I think salt concentration affects electroporation, but there is so little amount of total DNA in my ligation mixture. Do you still purify your ligation before electroporation then? I suppose there won't be much DNA left then.
As for heat shock transformation, 1~2ul of heat killed-but not purified ligation in less than 10ng of total DNA has usually been fine for me.
Can you also suggest some common electro comp cells? I think I have BL21(DE3).

Edited by Ecoli0157, 20 February 2009 - 08:50 AM.


#10 HomeBrew

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Posted 20 February 2009 - 04:49 PM

I have never seen the need to purchase competent cells -- the only time I did so was one time when I needed the strain, and the cheapest way to get it was to buy the cells competent from a commercial source. Companies don't have a magic wand; they use the same protocols you would to prepare competent cells. -- you can make competent cells in the lab for a fraction of the cost.




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