cloning of 40 nucleotide repeat fragments - (Jan/24/2008 )

Hi,
i'm planning to start some live cell imaging and for that, I supposedly need a MS2 ligand clone that has a {RNA leader-- 40 nucleotide ligand-- Terminator}. An imaging expert has told me that if I could clone 4-6 times the 40 nucleotide ligand between the RNA leader and Terminator I would get a better signal. I have no idea how to clone this 40 nucleotide ligand repeatedly. Could someone help me with this.

Thanks a lot

hepatitix

Hey there,

you can design a primer pair which contains your sequence (40bp, you could even design for 80 bp (2x40bp)) and sites for restriction enzymes which have complemetary sites (like SalI/XhoI). You anneal first the primers first and then insert in a plasmid. In the plasmid you have already two repeats now. Then you cut the plasmid with f. eks. XhoI and another enzyme (3.)which is in MCS and next to XhoI. The same plasmid you digest with SalI/the 3. to cut out your sequence. And you ligate them together. So, XhoI site will ligate to the SalI site and the 3. sites to the 3. site. the plasmid would contain 4x40bp.

Hi Zek
Thanks, I got the idea. That was very helpful.

You are wellcome:)

Watch out for Sal I - it can be a painful enzyme. Also avoid the EcoRI-Mfe I combo - Mfe I is not stable and EcoRI can problematic at times. So choose the combo wisely, Xba I-Nhe I would be fine.