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Sticky and blunt end ligation in single reaction?

Sticky end Blunt end ligation cloning

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2 replies to this topic

#1 Epigeneticist

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Posted 09 December 2014 - 03:59 PM

I only have two possible restriction sites for cloning an insert into my vector. One end is a sticky end (EcoRI) and the other end is a blunt end (PmeI or MssI). I have never done a blunt end ligation. Can I ligate my insert in a single reaction with these two different type of restriction sites? Are the reaction conditions for sticky and blunt ends ligation compatible for single reaction? Has anyone done this before? Or would this combination make my insert incompatible using these two restriction sites? 

 

Thanks!



#2 Rsm

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Posted 16 December 2014 - 06:49 AM

Yes, you can insert your gene using EcoR1/Pme1 in  a single reaction. I regularly do ligations using BamH1/Hpa1 (sticky/blunt), no problems.


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#3 Epigeneticist

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Posted 16 December 2014 - 08:52 PM

Thanks Rsm. I'll give it a try. Is the ligase reaction the same as sticky end or do you treat it like a blunt end reaction? Never done any blunt end cloning but I think you increase the amount of ligase and maybe add peg?





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