problem with ligation of linker/adaptor - (Apr/04/2013 )
I have designed 5'phosphorylated linker with sticky end or restriction sites. I have 25 nmol conc of each separate strand.
here is the sample consturct
5 ....agctt---------18bp----------gcggccgcac... 3
HindIII site 3.............a---------18bp-------gccggcgtgagct....5 xhoI site
I would really appreciate if you could tell me the followings:
1. What should be the solvent of these linkers? water or some buffer?
2. how should I anneal these strands together?
3. what should be the appropriate ratio of double digested vector and annealed linker
4. is concatemer formation is possible in this case?
1. I would do everything in TE. You need very small amounts of the annealed product in your ligation, so the TE will have little effect on downstream reactions, but will protect your DNA.
2. Heat a beaker of water to near boiling, put a sealed tube (best screw cap) with your DNA in it, and let it cool to room temperature. Adding 100 mM final NaCl will assist annealing. IDT sells "duplexing buffer" and has a protocol.
3. 1-3 moles of linker per mole of vector. This means you will need to highly dilute your linker, because there is too much in any sensible volume. Dilute in TE without salt.
4. Yes, the linkers can make a three component back to back concatamer. You can reduce the probability of this by lowering the concentration of your reaction, which will favor circularization prior to concatamer formation. This also argues for being on the low side of the linker concentration relative to vector.
Don't forget to phosphorylate the oligos (either order a 5' phosphate or treat with PNK).
Thank you Veteran