one blunt and one sticky ligation...something wrong - HELP!!! (Sep/18/2006 )
i am cutting my vector with spe I, purifying with qiagen kit, blunting using klenow fragment, purifying using qiagen kits, then cutting with Kpn I.
simulataneously, i cut insert out with ecorv, purify with kit, cut using Kpn I.
i ligate 3:1 ratio o/n 15C.
but my transformation produces only vectors.
does anyone have any suggestions?
-bwelsh-
QUOTE (bwelsh @ Sep 18 2006, 11:35 PM)
i am cutting my vector with spe I, purifying with qiagen kit, blunting using klenow fragment, purifying using qiagen kits, then cutting with Kpn I.
simulataneously, i cut insert out with ecorv, purify with kit, cut using Kpn I.
i ligate 3:1 ratio o/n 15C.
but my transformation produces only vectors.
does anyone have any suggestions?
simulataneously, i cut insert out with ecorv, purify with kit, cut using Kpn I.
i ligate 3:1 ratio o/n 15C.
but my transformation produces only vectors.
does anyone have any suggestions?
u may remove the phosphates on the 5' ends of your vector via CIAP reaction. this will highly eliminate vector self ligation products during ligation reaction. therefore it will increase the efficiency of your ligation reaction. u may also try a higher i:v ratio like 5:1
-dodosko-
Just to be sure... after cutting with kpnI, did you check that the digest worked?
Did you purify the products after kpnI digestion? If you didn't purify after the final digest the restricton digest buffer may not be optimal for the ligation reaction.
-ucsdgrad88-