religation of vector DNA

Anthony Tomlinson anthony at pplros.demon.co.uk
Fri Jul 29 05:39:27 EST 1994


In article <319a0a$6mm at news.doit.wisc.edu> Frank Anthony Skraly,
skralyf at cae.wisc.edu writes:
>In article <316d8p$ju1 at mserv1.dl.ac.uk>, Nelson Salazar
<N.SALAZAR at lshtm.ac.uk> writes:
>|> 
>|> Hello there,
>|>                 I am trying to clone two SacII DNA fragments (about 
>|> 6.5 and 7.5 kb in size) into pBluescript II separately, and I am not 
>|> getting recombinant plasmid after the transformation. I get many 
>|> colonies but when I screen them, they DO NOT have the insert DNAs 
>|> that I want, that is, the plasmid religates and gives me "false" 
>|> transformants.
>|>  
>|> How could I improve the cloning of my inserts ?? and how could I 
>|> avoid religation of my vector? I think that in this case 
>|> dephosphorylating of the pBluescript SacII cut does not make any 
>|> sense ? Any suggestions will be greatly appretiated.
>
>Did you ever try cutting after ligation with a restriction enzyme having
a 
>site in the insert but not in the vector (if such a site exists)? 
>
>Sometimes you have to buy a weird enzyme, but this always sharply reduces
>background for me.
>
>F.S.

Sorry to answer a question with a question, but I have two:
1)  Why is phosphatasing not a good idea?  
2)  If, after the ligation,  you cut with an enzyme which cuts in the
insert but not the vector, wouldn't you just linearise the vector-insert
molecules while leaving the reclosed vectors alone (ie the opposite to
what you want)?
Well okay three then (nobody expects the Spanish Inquisition) Are you
using a blue-white screen, and if so, and the whites that you pick are
all empty pBS, have you sequenced across the SacII site?  It could be
that in preparing your pBS/SacII, the ends have become chewed, thus
preventing the SacII insert from ligating in, but still giving whites
(because the LacZ is out of frame). IMH,  pBS does give 5 - 10% whites as
background, but anything above this is curious.

Anthony Tomlinson.



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