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"
>|>>|> 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
>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.
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.