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Making DNA Linkers for cloning purposes


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#1 Druss00

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Posted 23 August 2012 - 08:58 AM

I want to know if anyone has tried this. Design Oligos that will have 3' or 5' overhangs that correspond to a endonuclease restriction sites (I am trying to avoid cutting these linkers, so I thought I could engineer them already "cut"). I would then Ligate them using T4 ligase to phosphylate it. Will this work?

Thanks!

#2 bob1

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Posted 23 August 2012 - 12:40 PM

Yes, this is done - you need to get the HPLC purified option for your oligos so that you don't have all the shorter products that are found in normal oligo mixes. You also need to work out an annealing strategy to get double stranded DNA, basically this is resuspend the oligos in a buffer, heat to 95 C for 5 min, then cool slowly to room temp over an hour or so.

If these linkers are for adding to longer products then the ligation is relatively inefficient so you may need largeish scale preparations. Check out Sambrook's "Molecular Cloning:a laboratory manual" for methods.




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