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Ligation question, no clones after transformation

Ligation question, no clones after transformation - Molecular Cloning Forum

Ligation question, no clones after transformation - DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


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Old 08-06-2010, 05:06 PM
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Default Ligation question, no clones after transformation



Hi guys,
I had a problem when trying to clone a 1.8kb insert into a 8.3kb vector. I first digested both insert and vector and purfied from the GEL. Then I did a rapid ligation and transformation afterwards. But I can not even see a single clone.
One thing to add: I checked the ligation on the GEL. I can see negative control (no ligase) with clear two bands. For others, I can see smear bands with larger size (bigger than the insert + vector). [see attached pics]
I speculate that this ligation might works but not properly, but I do not know the possible reason.
I would like to ask you: if the ligation is successful, what band would you see on the GEL? Is there something wrong if I see smear bands with unexpected larger size?
tony
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Old 08-12-2010, 11:02 AM
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Default Re: Ligation question, no clones after transformation

Remember that when you have ligated your insert into the vector it will behave like closed circular DNA, therfore will not migrate on the gel as you would expect linear DNA to. Maybe you do actually have a ligated product. What ligase are you using? are all the buffers in date? remember to quantify your DNA and vector and use a range of ratios for ligation to ensure you get the optimum chance of ligation
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Old 08-12-2010, 02:20 PM
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Default Re: Ligation question, no clones after transformation

thanks for your reply, hcarre.
I am using T4 ligase from Roche rapid ligation kit. The kit should be fine and is far away from the expire date. I tried ratios (insert: vector) such as 3:1, 5:1. None of them had clones grow.
As you mentioned, the ligated DNA will be closed circular. So it should be running faster in the GEL than linear ones, right? I rember last time of my successful ligation, the lane for ligated DNA is almost nothing rather than a large band.
Maybe I put too much DNA for ligation. Is is problematic for ligating 160 ng insert and 200 ng vector?
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Old 08-14-2010, 02:18 AM
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Default Re: Ligation question, no clones after transformation

It would be good if total insert + vector = 100ng in 10ul reaction.
Also keeping the reaction mix at 12 degrees overnight might help.

Last edited by rasing02; 08-14-2010 at 02:23 AM.
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