Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Cloning

Blunt ligation with PCR: is kinase needed? - is it necessary to phosphorylate if the vector is not CIAPed? (Apr/24/2009 )

Pages: Previous 1 2 

lna on Apr 27 2009, 06:38 AM said:

perneseblue on Apr 27 2009, 01:00 PM said:

lna on Apr 26 2009, 11:58 PM said:

mastermi on Apr 24 2009, 06:04 PM said:

Why don't you use primers with NotI sites, cut the PCR product and make a sticky end ligation?

I just did not wanted to order the additional primers. moreover, I've already tried to do the ligation of this sequence with another restriction sites-containing primers (into another vector) and it was not working (do no know why).


Was the NotI site skirted with enough bp? NotI site requires a minimum of 6bp before it will be cut efficiently


it was not the Not site that I used before (because I was trying to insert into another vector). I used BamHI and XbaI and I left 4 additional nt on the ends - to my knowledge this should be enough for these enzymes..
Thank you for your interest. Actually, I was trying to verify here, whether the kinase reaction is needed to ligate the PCR product into the cut vector.


I have not used kinase throughout my time doing cloning. u only need one phosphorylated end on both side. and then the E.coli have mechanism to gap repair the unligated portion of it.



P-------OH
OH-----P

after ligation

----OH-P-------OH OH-------
----OH OH------P-OH--------

after E.coli

----OH-P-------OH-P-------
----P-OH------P-OH--------

-hanming86-

hanming86 on Apr 29 2009, 05:24 PM said:

lna on Apr 27 2009, 06:38 AM said:

perneseblue on Apr 27 2009, 01:00 PM said:

lna on Apr 26 2009, 11:58 PM said:

mastermi on Apr 24 2009, 06:04 PM said:

Why don't you use primers with NotI sites, cut the PCR product and make a sticky end ligation?

I just did not wanted to order the additional primers. moreover, I've already tried to do the ligation of this sequence with another restriction sites-containing primers (into another vector) and it was not working (do no know why).


Was the NotI site skirted with enough bp? NotI site requires a minimum of 6bp before it will be cut efficiently


it was not the Not site that I used before (because I was trying to insert into another vector). I used BamHI and XbaI and I left 4 additional nt on the ends - to my knowledge this should be enough for these enzymes..
Thank you for your interest. Actually, I was trying to verify here, whether the kinase reaction is needed to ligate the PCR product into the cut vector.


I have not used kinase throughout my time doing cloning. u only need one phosphorylated end on both side. and then the E.coli have mechanism to gap repair the unligated portion of it.



P-------OH
OH-----P

after ligation

----OH-P-------OH OH-------
----OH OH------P-OH--------

after E.coli

----OH-P-------OH-P-------
----P-OH------P-OH--------


thank you! I guess, this answers my question directly! (:

-lna-
Pages: Previous 1 2