ligation step in cosmid library construction - (Jul/17/2007 )

Dear All,
Now, I am doing fungal cosmid library construction. Unfortunately, I can not pass the step of ligation. I use Super Cos1 cosmid vector (Stratagene) as the vector and insert fragment is 35 - 40 kb fungal genome partial digested by Sau3AI.
I have changed ligase enzyme (with or without 10 mM ATP) also the time for ligation. Moreover, I try to do self ligation of cut vector. I found that it can be self ligation but the efficient of self ligation is high. So, I dont know that which step is wrong or have the problem.
Could you suggest me about the construction method or another kit? Otherwise, Please suggest me about the tip in cosmid library construction.

Thank you very muck
Nay

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How are you transforming the cells? Efficiency for transforming large plasmids is low for chemical transformation. Are you dephosphorylating the cut vector? How do you know the ligations are not working?

Short version: tell us exactly what you are doing and what the results are. We can't help without knowing this.

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Thank you very much for your answer.
This is the detailed of my protocol. Firstly, I do partial digestion with entomopathogenic fungus by Sau3AI and dephosphorylate it. For preparing the plasmid, Superocos1 cosmid was digested by XbaI and puirifed by phenol CH3Cl. Next, I do dephosphorylate of XbaI - digested vector and purified by phenol-CH3Cl. Finally I digest the XbaI-CIAP vector with BamHI and purified again by phenol-CH3Cl.
In the ligation step, I use T4 DNA ligase to do ligation. I take 1 ul of the sample before add ligase to check for background by add every reagent same except no add partial digested genomic DNA and ligase enzyme. After ligation at 16C o/n, I check the ligation reaction using southern analysis using the part of SuperCos1 as probe.
But from Southern analysis result, I can not see the shift band to high molecular weight. The position of sample the sample before and after add enzyme is same molecular weight. So thats mean the ligatin isnot successful. My genomic cant ligate with the plasmid.
So, I stop at this step (dont continue to do packing). I already change many conditions for example ratio between DNA and vecor, type of ligase enzyme, temp. and time for ligation, method for DNA extration but the all results are same. No ligation.
Could you suggest me what I should do to solve this problem or I mistake in some step.

Thank you again
Nay

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if I read this correctly, both your insert and vector are dephosphorylated. If that is the case, not only is the vector unable to ligate to itself, the insert too can not ligate to the vector.

More over, the XbaI and Sau3AI sites are not compatible. They do not ligate. So if I read this correctly, only one end of the cosmid could ever ligate with the insert.

Finally, the current method of sequentially dephosphorylating the 2 overhangs, will result in the first overhang (the XbaI site) to become overdephosphorylated. CIP is a bad enzyme and will damage your ends if over used.

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