Normally I was using 1 uM and it was ok.

What is the melting temperature of your oligos and predicted secondary structure ?

Thanks Dawid for the answer. Can you tell me 1uM concentration for each ss oligo in how much volume of annealing buffer. I am planning to use 10mM Tris-HCl as my annealing buffer and not use EDTA as I want to perform ligation with a vector. I am also thinking of adding NaCl and MgCl2 as I read it on internet that some people were using it. Once they get annealed how much should be the insert to vector ratio.

As for the melting temperature I used generunner to analyze my oligos and found that one had Tm around 30 while other had around 70. But as I am going to denature it by heating to 100 degrees I guess it should not affect.

Yes, I used 1 uM each in 30 ul (it gives 1 uM dsDNA).

If it is about the buffer you can use plain water or (as I did) ligase buffer, so you will have no problem while ligation.

The insert-vector ratio might be 3:1, 5:1 oraz higher. It should not be that big problem.

Thanks alot David. I will try your suggestions. I have got PAGE purified oligos that I am afraid if I don't get the annealed oligos the first time I will be in big problem. Anyways I will try and thanks for the tip.

I am going to prepare 100uM stock after resuspending the oligos and then take around 0.3uL to get 1uM in 30uL. This makes things easier for me as I do have very less amount of oligos. Thanks alot. I will let u know wen I get positive results. Bye.:)

I have one more question. Do I need to dilute the 1uM oligos for ligation or can directly go ahead with it?

You should dilute it. As I said before you should have molar ration insert vector~3:1 during ligation.

http://oomyceteworld.net/protocols/LigationCloning.pdf

I think you can find most of information you need there.

Ya I understood how to go about it. Thanks again for taking out time and answering my doubts. Have a good day, bye

Are you trying to clone for shRNA ?? then I would suggest go for Gradual cooling and then you can check your annealing by running 4% gel with annealed & single stranded primers. Also I used 10 microM and it worked well for me .

Thanks Disha, I am trying to anneal these two oligos to ligate with my bacterial expression vector, not going for shRNA though. Do you mean 4 percent of agarose gel? coz i read somewhere they ask to run on acrylamide gels as well.

Yup 4% of agarose gel. I did similar sized annealed primers cloning but was for shRNA & for me concentration of Primers created lot of nuisance in the beginning but when I did with diluted primers I'm getting good number of positive colonies.

Did you anneal your dsoligo with a vector? If I have understood you correct then you took 10uM of ssoligo in some amount of annealing buffer and put it for heating then when they got annealed u checked on gel, then diluted this ds oligo according to vector: insert ratio for ligation?

You understood it correct, I did gradual cooling in PCR for annealing than diluted it. I took 50 ng of vector +2-3 uL of Annealed Primer( diluted) + 1uL of Ligase buffer + 1uL of T4 DNA Ligase , this was suggested in many papers & this worked perfectly well

Thanks alot Disha, if I have any doubts while doing it I will ask you. Have a great day:)

Hello, I will do the same experiment and I have a question! The 2 oligos need to be 5-phosphorylated prior ligation? I think they only have to be 5-phosphorylated if the vector is dephosphorylated, right?