Restriction near DNA ends

Martin Kennedy cytogen at chmeds.ac.nz
Thu Dec 10 01:38:32 EST 1992


In article <01GS494N2CK28WW1OD at mis.mcw.edu>, DMEIER at mis.mcw.edu writes:
> Hello all!
>   I'm preparing to do a PCR and I've had some very good suggestions from 
> this group in terms of how to sequence the PCR product.

	--stuff deleted--

>   Can someone please send a small list of restriction enzymes they know 
> will cut near a DNA end, and what is the recommended amount of spacer DNA 
> to add to the end.
>   Thanks very much in advance for your advice.
> 
>   Dan Meier
>   DMEIER at mis.mcw.edu


I always make my PCR primers with a 3 base add-on 5' to the restriction site;
eg for an EcoR1 site I do: 5'-TAGGAATTC....3'.  I use the trinucleotide TAG at
the 5' end, but any three bases should do.  I've cloned dozens of PCR fragments
in this way with combinations of XhoI, EcoR1, HindIII and BamH1 ends. 

Cheers,

MArtin



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