A short linker

[Troels Wind]

Yoram Gerchman (yoramg2 at LEONARDO.LS.HUJI.AC.IL) wrote:
: Greetings. I'm tryng to insert a short linker (about 45 nt) into a
: restriction site in the middle of a gene. Now, my idea was to creat the
: linker in such a way that it will leave cohesive ends that fit the RE.

: somthing like

: 	AAACCCCCCCCCCCC	
: 	   ||||||||||||
: 	   GGGGGGGGGGGGTTT

: Anyone tried this?
: .........................................................................
: Yoram Gerchman
: Division of Microbial & Molecular Ecology
: The Institute of Life Sciences
: Hebrew University of Jerusalem
: Givat Ram, Jerusalem 91904, Israel
: Tel: 972-2-6585175

What you suggest is doable, but look out for the following:

1)	Dephosphorylate the vector after cutting to avoid self-ligation.
2)	Make sure that your oligoes (the linker) is 5'-phosphorylated. You
	can do this yourself or probably make the manufacturer of the oligoes
	do it.
3)	Look out for multiple insertions! Since you use only one RE-site,
	you risk that several linkers will be inserted as repeats. Thus,
	perform ligations with different vector:insert ratios, the higher
	ratio the better the chance of a single linker insertion.
4)	To save yourself some sequencing, look carefully for RE's whos
	digestion-pattern can reveal if you have multiple insertions.
	For example, if RE XxxI cuts on both sides of the site that you
	clone your linker into giving a fragment of approx. 500 bp,
	you can easily screen your clones with XxxI and identify those
	with a single linker inserted (positive clones will then give
	a 545 bp band).
5)	Your linker may enter the RE site in both directions!   

Good luck!

/Troels