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about ligation and transformation


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#1 lyrezxl

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Posted 29 August 2003 - 05:27 AM

Dear all,

i never get a terrible trouble like this. it's a normal ligation and transformation. the vector is 13kb and the insert is 3kb, be used in HindIII site. i have transforme several times, and there is no any clone on plate. the efficiency is about 10-7/ug.
i have designed severy controls: the small clone vector digested by EcoRI, the self-ligation, w/wo dephosphorylation, another one is the vector added the insert as the v/i rate 1:3. at last the self-ligation is normal but when i added the insert, there is no clones.
even i transformation by electro-transformation.
i have no idea now.
anyone can help me?
thanks a lot

lyre

#2 sergechampetier

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Posted 29 August 2003 - 09:45 AM

I have had the same problem a couple of years ago. I solved it after several frustrating weeks of unsuccessful work. One thing most people are not aware of is that commercial kits for purification of DNA bands from gel either introduce or fail to get rid of contaminants which inhibit the ligation/cloning process; this effect frequently goes unnoticed with very efficient reactions such as cloning of regular fragments (1-3kb), but with longer DNA fragments (more that 10kb) this becomes a major problem. Unfortunately I can't remember the exact method I switched to with instant success, but it involved traditional phenol (CHCL3?) extraction steps INSTEAD OF commercial kits. Incidently a paper was published several years ago by people who compared the cloning efficiency of six methods for purification from gel of large DNA fragments, with the conclusion that the phenol method was the best, but again I regret I don't remember the exact reference. Good luck anyway.
Serge Champetier Ph.D.
Quebec City.

#3 lyrezxl

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Posted 30 August 2003 - 04:22 PM

Serge Champetier Ph.D. Thanks a lot.
l never used the method by phenol (CHCL3?) extraction, and l don't know how to do it. but in our lab, we also use the glass wool or something like it to recove the DNA bands.
sometimes we also precipitate the ligation solution. l think l should try it.

#4 vgupta

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Posted 02 September 2003 - 02:20 PM

yes, glasss woool works really good. i have had the same problem. While purifying the gel piece by qiagen kit, i wash the column three times instead of once or twice.




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