Dephosphorylation of vector before ligation - Is it required? (Jan/26/2005 )
so my Q is how do u go abt digestion after ligation. do i have to heat kill ligase before i put my enzyme or i can directly perform on the mixture.
Mm I didn't knew that. I'm using the Invitrogen T4 ligase and it doesn't say anything about heat inactivation. That's interesting. I'll try that next time, 'cause I usually inactivate it.
When I digest after ligation what I do is drop dialisis the ligation, you know, using Millipore membrane filters, for half-an-hour, and then add the appropriate RE buffer and the enzyme. I usually inactivate the ligase @ 65ºC for 10 min, but maybe this is not necessary, as changing the buffer and incubating at 37ºC may decrease a lot its activity. T4 ligase is unstable at high temperatures, and salt inhibits it. Cheers!
Hi Badcell,
well good news finally. i got 0 colonies in my no-insert control ligation plate. and around 30-40 colonies in my other transformant plates. ur suggestion worked real goood!! i like to mention tht i did not heat inactivate the ligate mixture. i added 0.5ul of rest enz+10ul enz buffer. added 1ul to the ligation mixture and incubated for 15mins at 37C. then transformed.
its better tht u too try without deactivation liagase as u may also get 30 colonies instead of 3!!! good for u. u saved my day.
cheeeeeeeers.
rajgene
Hi! Glad it went so well, rajgene 
Next time I'll be sure to try without deactivating the ligase. Cheers!
One useful trick when cloning using two different sites is checking if you loose any site in the vector multiple cloning site (if any site is between the two REs you are using and not anywhere else in the vector). If this is the case, and this site is not found neither in your insert, you can digest your ligation with that enzyme and you will cut only your religated plasmids and not the ones with your insert, which will not have that site. So you will get only colonies with the insert.
Badcell-
that is indeed a great trick and would like to use it..but how do you know how much enzyme to add to the ligation..do you just base it on how much DNA you used in the ligation? And waht about matching enzyme buffer rxn conditions?
Slapolla
Hi slapolla
I don't really calculate the quantity of ER I add depending on the DNA used. I usually add 0.5 ul of ER and incubate for several hours (2-3 h, although sometimes I leave it o/n for convenience). This is more than enough, as your DNA would be on the ng range. Probably 1h of incubation will do it.
As for the buffer conditions, I sometimes dialyze the ligation for half an hour using spot dialysis filters from Millipore, and then I add the corresponding ER buffer and the enzyme. But I've also tried it without dialysis and it works. You can add directly the 10Xbuffer to the ligation mix, and the ER... and cross your fingers. For me this little trick really works.
Good luck
Thanks so much badcell! When I have a construct that I can use that trick for I will try it and let you know how it goes.
Hi,
I agree that the concept is clever.
However, all considering, isn't it just as easy to dephosphorylate - just in case? Using SAP, you can add 5 Units (let's say) the last 15 min of your RE incubation, then heat inactivate. This should take care of all your single cut plasmids.
I'm not a big fan of dephosphorylation, as SAP has munched away my DNA several times, and I know from other people that this can happen quite often. Also, dephosphorylation, when it works, is not 100% efficient. Just as after overnight incubation with an RE not all your plasmid molecules would have been cut, after the much shorter 15 min incubation with SAP/CIP/wathever, not all your digested molecules would be dephosphorylated. After saying that, I must say that I usually dephosphorylate my vector DNA extensively, for 30 min-1h, but I always keep an aliquot untreated... just in case SAP is hungry.
Cheers
wow.. im in highschool and i have no idea what anyone is talking about. haha. it sounds outrageously intresting though
[FONT=Arial][SIZE=1][COLOR=green]wow.. im in highschool and i have no idea what anyone is talking about. haha. it sounds outrageously intresting though
hi everyone!i find these little tricks interesting. i want to ligate a 600bp insert into a 5.1kb vector, cut by single rest. enzyme(Bam HI).i do not get the insert-vector ligate and after carrying out digestion of ligation mix i only get a smear!can anyone help me out! the insert plasmid is of low copy number.is it necessary to carry out dephosphorylation before ligation?