How do I confirm whether the ligation has occurred? Is there any method to confirm whether the ligation has happened, using the ligated product, other than to check with antibiotic selection post transformation?
You can do PCR with universal primer as mentioned by Nasrin using ligated and un-ligated product as a template and then compare PCR product size between these two products.
Thank you Nasrin and Chaiyaporn, but I wanted to confirm before plating or picking colonies. I have tried cloning by ligation and there are no colonies in the plate. I was just wondering if there is something I could do one step before with just the ligated product - soon after overnight ligation reaction - (just to see if has really ligated or if the cloning is failing because of improper ligation)
Hi Joel, you can keep a negative control where you only plate your double digested product. In ideal conditions, it should not grow and also it's a double digestion so no chances for self ligation as well. But if you want to validate whether or not your ligation is successful before plating, you can single digest the lighted product and run it along with your uncut plasmid and double digested plasmid in 1% gel. If you run it along with 1Kb and 100bp ladder you can understand clearly whether your product is lighted or not. Your double digested plasmid will show 2 bands whereas your single digested plasmid will show 1 band. And also your uncut plasmid will be as it is how a plasmid looks like. Hope this helps.!
Thanks Roy. I had the same strategy in mind, but was just wondering if the ligated product can be used as a template (as there are buffers in the same milieu as the ligated product)
I think you can use it. That is the same concept as colony PCR only you are doing the PCR before transformation. That's it right! ?
No No. If it is not ligated and you haven't transformed it yet, then also your insert will get amplified. I don't think that's a good move.
Using a ligated product for PCR wouldn't generally work as there is a possibility of seeing too much non-specificity in the amplicon. As Arkaprava said, non-ligated amplicon also gives you a band and you still wouldnt know if your gene has been ligated in the plasmid. Best option would be to trasnform and perform a colony PCR than looking at the ligated product.
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