Phosphatase treatment of sticky ended products? - (Sep/15/2006 )
Hello all
Trying to insert an 800 bp fragment into a 2400 bp vector. Both were double digested with Bam HI and Hind III. The two were to be ligated using Roche's Rapid Ligation Kit (T4 ligase). I was having early problems so took it on myself to dephosphorylate bothe the digested vector and the digested 800 bp frag with calf intestinal and shrimp phosphateses.
I was told that although this was fine for blunt ended ligations, but for sticky ended ligation at least the vector or the insert has to have phosphates to allow the ligation to proceed. Before I start wasting more time on ligations and transformations, can anybody tell me if this is true or is an old wives tale??!!
I know that you can dephosphorilate the vector but I don't think you can dephosphorilare the insert as well: how can you ligate without a free phosphate?
yes, that's right
for a vector, dephosphorulation is important to avoid vector closure, but
for the insert, it's not since it's just a fragment
and according to this, the vector is what we have to work with for ligation,
therefore wheather the vector have blunt ends or sticky ends it should have a free 5-P at both ends...
let us wait to see other replies..
we dephosphorylate only the vector but thats only for blunt end cloning. For sticky ends, we rarely go thru this procedure.
If u dephosphorylate both the vector and insert, ligation will not work.
Thanks everyone,
The sticky ended ligation worked when neither the vector nor the insert were dephosphorylated. However the ligation did not work with a phosphatase treated vector and an untreated insert. Not sure why but I got it to work once so Im happy!!
Anyone know a good alternative for Novozyme 234 for protoplast formation?
not sure about the protoplast thing, but wanted to add my .02...
for the phosphatase, depending on which type you used and your reaction conditions, the enzyme can chew up the ends of your DNA fragment once it has removed the phosphate. so, sometimes it will not work because perhaps you have destroyed one or both of the ends such that the sites no longer exist and they are unable to religate back to a complementary sticky end. sometimes you have to play with the conditions of the phosphatase reaction in order to get the best dephosphorylation without degrading your DNA
for the phosphatase, depending on which type you used and your reaction conditions, the enzyme can chew up the ends of your DNA fragment once it has removed the phosphate. so, sometimes it will not work because perhaps you have destroyed one or both of the ends such that the sites no longer exist and they are unable to religate back to a complementary sticky end. sometimes you have to play with the conditions of the phosphatase reaction in order to get the best dephosphorylation without degrading your DNA
They didnt mention that in the manual!!! Used a combination of shrimp and calf intestinal alkaline phophates from Roche. Would explain why I got successful ligation when neither vector nor insert were dephosphorylated and an unsuccessful ligation when the vector was treated with phosphatase.
Thanks for the .02!!
I agree with aimikins -- deposphorylation is a balance between minimizing your chances of vector self-ligation and destroying your vector.
The experiment in question needed no dephosporylation at all, because (assuming both enzymes cut the vector), self-ligation of the vector was not possible. But, when you need to deposphorylate a vector due to a single enzyme digestion or blunt-ended digestion, use very little dephosphorylase, incubate the dephosphorylation reaction for a limited amount of time (5 to 15 minutes at 37°C), and gel-purify the vector before use.