TE buffer will be good.

Good Luck !!!

Hi,

usually, you have to phoshorylate the oligos in addition to annealing, too.

To do so, I dissolve the oligos in ddH2O (100 µM) first and run the following reaction:

Oligo top (100 µM): 1 µl

Oligo bottom (100 µM): 1 µl

10x T4 Ligase Buffer (Thermo Scientific): 1 µl

T4 Polynucleotide kinase (PNK, 10 U/µl): 1 µl

ddH2O: 6 µl

37 °C 30 min

95 °C 5 min

ramp down to 25 °C at 0.1 °C/sec

afterwards, I dilute the annealed and phosphorylated oligos 1:200 and use them for ligation. However, exact amount always depends on the size and amount of your vector.

Good luck,

Floriane

Thanks for the answers,

Floriane, Why you do annealing and ligation at the same time?

I thought they should be done separately. Also, as I have not done dephosporilation in the vector, I may not need to do phosphorilation for annealing stage. Am I right?

These dilutions are OK for any oligose with different MW? Or I should calculate the exact concentration of them? My oligose are having very high MW.

Many thanks,

Mah

Thanks for the answers,

Floriane, Why you do annealing and ligation at the same time?

I thought they should be done separately. Also, as I have not done dephosporilation in the vector, I may not need to do phosphorilation for annealing stage. Am I right?

These dilutions are OK for any oligose with different MW? Or I should calculate the exact concentration of them? My oligose are having very high MW.

Many thanks,

mah