ligation with sticky and blunt ends - (Mar/24/2006 )
Hi, guys:
I am trying to ligate a 4kb fragment (XbaI and HpaI) into a 9kb vector. But one end is sticky and the other is blunt. I do not know if I should follow the protocols for blunt end ligation? for example, should I use phosphotase and increase insert:vector ratio?
BTW, can I digest with HapI first, dephosphorylate, gel purify, then XbaI, gel purify. Finally ligation?
Really appreciate your help!
Suny
I recently did the same (double digest with EcoRV and SacII).
Here are the steps I did: double digest together (provided they can work in the same buffer), sap treat (in case there is any singly cut vector so that it doesn't ligate back together), gel extract, ligate with the usual protocol for sticky ends (3x molar ratio insert to vector, 14 degress overnight). I had no problems with this.
Alternatively what you can do if you want to avoid gel extraction and sap treatment altogether is make use of any restriction sites that are found in the part of the vector you plan on removing and that are not found in your insert. After ligation, digest with this enzyme to cut any religated or uncut vector. For example, if the part of the vector you are removing contains an EcoRI cut site, and this site is not found anywhere else on the vector and is not found on your insert, you can digest the ligation with EcoRI to remove the unwanted background.
Someone on this forum recently posted this idea and it sounds great (I can't find the post).
Cloning master--BadCell posted it
Thank you all. It sounds perfect. I will try it.
Here are the steps I did: double digest together (provided they can work in the same buffer), sap treat (in case there is any singly cut vector so that it doesn't ligate back together), gel extract, ligate with the usual protocol for sticky ends (3x molar ratio insert to vector, 14 degress overnight). I had no problems with this.
Alternatively what you can do if you want to avoid gel extraction and sap treatment altogether is make use of any restriction sites that are found in the part of the vector you plan on removing and that are not found in your insert. After ligation, digest with this enzyme to cut any religated or uncut vector. For example, if the part of the vector you are removing contains an EcoRI cut site, and this site is not found anywhere else on the vector and is not found on your insert, you can digest the ligation with EcoRI to remove the unwanted background.
Someone on this forum recently posted this idea and it sounds great (I can't find the post).