Palindrome and ligations

[John Dixon]

In article <3v2jrg$962 at apopi.u-strasbg.fr>, Francois Nantel
<Nantel at titus.u-strasbg.fr> wrote:


> ligation in which a 3.2 kb blunt-sticky fragment is inserted into a 4.8
> kb vector. Up to now, all I have are empty plates. I tried a lot of

> 
> In fact, this insert contains the loxP sequence very close to
> the edges. The loxP sequence is a 32 bp palindrome used for recombination
> experiments. I am wondering if such a large structure can interfere with
> the ligation reaction and, if so, how could I correct it?
> 

Hi Francois,

I have a vector with two tandem loxP sites separated by an XbaI site such
that the first T and final A of the Xba site (TctagA) are the end and
beginning of the two loxP's. I have had no (unusual) problems cloning into
here, both blunt-ended and stickily. 

Although they are partly palindromic ie 13bp inverted repeats flanking a
non-palindromic 8bp spacer, they should not be in an unusual conformation
in double stranded DNA.

So I doubt that your loxPs are causing the problem, but they themselves
may have a problem as they should be 34 bps long ;-)

For advice on sticky 1 end and blunt other, check out the smaI religation
thread elsewhere.

Good luck

-- 
John Dixon                                        Lab 44 (1223) 334131
Wellcome/CRC Institute                            Fax 44 (1223) 334134
Department of Genetics
Cambridge University    
United Kingdom                           e-m: jpcd0 at mole.bio.cam.ac.uk