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generating small DNA fragments with stick ends by annealing oligos?

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#1 donny



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Posted 01 March 2012 - 07:39 PM

I need to clone a very small DNA fragment encoding a small peptide (~35bp) into a vector with a fusion protein tag. I'm thinking since this is such a small fragment, I can design two oligos such that they will anneal and the sticky ends will be as desired. I can then clone the hybridised DNA into the vector. That way I can do away with digestion with restriction enzymes and avoid difficulties in purification since the lowest cutoff for spin columns is 40bp. Does this make sense? Has anyone tried this?

#2 Rsm


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Posted 02 March 2012 - 03:25 AM

Yes, that will work. People will tell you to phosphorylate the 5' ends of the oligos, but that's not necessary. Use different/incompatible ends for this, otherwise you'll have many repeats. The cutoff for spin coumns is 70bp, if you want to go lower, you can use QIAEXII or Etachinmate.
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