The DNA fragments have been previously digested with restriction enzymes, will ligation after ethanol precipitation work?
Yes, for sure they will work. But you need to dry the precipitate at room temperature (25-30 ºC) for 15-20 min and dissolve in nuclease free water at room temperature for 15-20 min. Do not warm/heat for dissolving. Over-drying may create problem in solubility when you dissolve the precipitate in water after drying. Traces of ethanol/isopropanol (incomplete drying) in the precipitate may create problem for further downstream reactions. Make sure that you have removed ethanol/isopropanol completely before you keep for drying. The precipitate looks opaque white when not dried completely and looks translucent when dried completely. It will be more better if you run the DNA fragment in agarose gel, elute it and then proceed for ligation. Good Luck !!!
Of course¡¡¡ you must wash it wtih EtOH 70%, remove the EtOH, dry it (air/10' or 37ºC/ 3') ans then resuspend in Molecular Biology grade water)
Yes, for sure they will work. But you need to dry the precipitate at room temperature (25-30 ºC) for 15-20 min and dissolve in nuclease free water at room temperature for 15-20 min. Do not warm/heat for dissolving. Over-drying may create problem in solubility when you dissolve the precipitate in water after drying. Traces of ethanol/isopropanol (incomplete drying) in the precipitate may create problem for further downstream reactions. Make sure that you have removed ethanol/isopropanol completely before you keep for drying. The precipitate looks opaque white when not dried completely and looks translucent when dried completely. It will be more better if you run the DNA fragment in agarose gel, elute it and then proceed for ligation. Good Luck !!!
yes, but it may depend on the compatibility of the ends which the restriction enzymes created. There also is a way of blunt end cloning which is more challenging, but routine since decades - and current protocols should be available almost everywhere.
Dear Dr. Robert H. Eibl,
I found your answer interesting. For the sake of my interest, I would like to request you to kindly comprehend your view regarding the compatibility of digested ends. Are you referring to sticky and blunt end ligation?
Vielen Danke.
It will definitely work. no need to worry as all the above say, ethanol/isopropanol must be dried completely. This kind of work is needed when one wants to do a double digestion and the enzymes cannot be deactivated......
Yes you can. Forget about what all other said. Just clean these (wash) and ligate. It works!
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