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