ligation--pls help! - (Jan/05/2011 )
hi all,
after ligation of my vector and insert, when i run it on the gel, what exactly should it look like? should it run at the exact size that my clone is supposed to run? should it be a single band? can someone pls help me on this, since i am getting bands of high molecular weight, high above the ladder, near the wells. but transformation is not happening, i tried different inserts and vectors, did every possible thing like increasing/decreasing concentration, making new plasmid and insert from scratch a few times and restricting them all over, etc. my competent cells are perfect since my controls are perfect:( plss help.!
When you say that your controls are perfect, does this mean you have measured the competence of your cells? What is it? This is certainly the most common cause of failure. Are you using quick ligase or normal ligase buffer? Do you heat kill the ligase before running the gel? You may also have a construct which is lethal to the cells, so that even if the ligation is correct, the transformation does not work. We need a LOT more detail to begin to try guesses at the problem.
I may recommend the following website for very important tips. They really saved a lot of time for my ligations, time, and reagents.
http://bitesizebio.com/2007/11/20/pin-pointing-dna-ligation-problems/
I am trying to clone a 2kb microRNA promoter gene and its deletion constructs into a luciferase reporter. After ligation,i transform 4ul of ligation mix in 100ul of cells and get no colonies. I use NEB quick ligase and ligate for 10mins at 25C. If the ligation mix is run on the gel, i get a very high molecular weight band, for any insert and any vector. Is this the ligated plasmid's band? i never get it in the expected size. I always get it way above.
i do not heat kill ligase before running on the gel.
Unless you are doing blunt ligations, I would switch to normal ligase buffer instead of the quick ligase. The PEG in quick ligase makes the gel give very high bands, and can't be heat killed. Normal ligase buffer can be heat killed and gives normal DNA bands, which can be used to analyze the ligation products. Usually, these are too complex to provide much insight, however, unless you are doing controls with carefully designed single-fragment self-ligations.
In my ligation experiments, I saw bands like aggregating to the size of the vector used (e.g pUC19/pUC18 etc.) It looks like they are forming a ladder in the picture. I think you are doing okay.
phage434 on Fri Jan 7 01:14:34 2011 said:
Unless you are doing blunt ligations, I would switch to normal ligase buffer instead of the quick ligase. The PEG in quick ligase makes the gel give very high bands, and can't be heat killed. Normal ligase buffer can be heat killed and gives normal DNA bands, which can be used to analyze the ligation products. Usually, these are too complex to provide much insight, however, unless you are doing controls with carefully designed single-fragment self-ligations.
But have you faced this problem of very high bands? It gives the same sorta band for any ligation i do but ultimately transformation is not working. I do not get colonies. my positive and negative controls are giving the expected colonies. I do not know where else i am going wrong. pls help!
pls help 
Electroporation with quick ligase do not work. PEG in quick ligase inhibit the electroporation. Try heat shock if you are using quick ligase.
Or switch over to regular T4 ligase and then use electroporation. You can also transform with Z.comp cells i.e. 5 minute transformation using regular T4 ligase.