Ligation failure using three DNA fragments. - Cohesive ligation failure (Jun/19/2006 )

Hi,

I work on neuromuscular disorders. My intention is to ligate three linear DNA fragments. The fragments are of length 242bp, 1.8 kb and 485 bp respectively. My ligation should be in such a way that the 1.8 kb fragment is between the other fragments, the orientation is not a matter since it is a spacing sequence. The 242 bp fragment has a Not I site in the 3" end. The 1.8 kb fragment had Not I site in both ends and the 485 bp fragment has NotI site in the 5" end.

I did a NotI digest of the three fragments and tried to ligate them using Quick Ligation Kit from New England Biolabs.

After ligation, I tried to do PCR amplification of the 242 bp+1.8 kb+485 bp ligated fragments using the forward primer of 242 bp fragment and the reverse primer for the 485 bp fragment. I do not get the required product. Instead I get something like 500 bp product.

I also tried doing ligations using two fragments at a time and then doing PCR but that does not work. I also tried to increase the time of the ligation without success.

My PCR conditions are : 94 degrees -------------------- 3 minutes
94 degrees -------------------- 1 minutes I
50 degrees -------------------- 2 minutes I---------- 30 cycles
72 degrees -------------------- 1 minutes I
72 degrees -------------------- 10 minutes
4 degrees -------------------- forever.
End.

I do not know how to make it a successful ligation. Please let me know if there is any modification needed in the ligation step or the PCR conditions.

With warm regards,

Coolgene

hi,
linear DNA pieces can usually generate concatemers. your 500bp fragment seems to result from concatemerization. you should dephosphorylate one of your fragments or may be two of them so you will decrease the probability of having concatemers. as far as i understand, you need to dephosphorylate both 485bp and 242bp fragments. then your ligation reaction will be led by the p's on 1.8b fragment.
i generally use CIAP for dephosphorylation but i cannot say it is efficient. somebody else may recommend you some other enzymes hopefully

U have to increase the elongation time from 1min. in ur PCR to something like 2.5min bcoz the entire length which ur trying to amplify is nearly 2.5kb. i calculate 1kb per min for elongation.

Hi,
I’m trying to ligate by PCR too.
I need to ligate a vector digested with PacI, and two 1.5 fragments digested with PacI-EcoRI, I tried many times without good results. I tried different rations 1:1:1, 1:3:3, 1:6:6. I tried dephosphorilation of my vector, but if I do so, I have no colonies!! (just a little of background). I tried to ligate the two fragments by PCR. Just ligating fragments and using this reaction in a PCR reaction with no results. Do you think I need to precipitate before PCR? How much fragments do I have to ligate. And How much of my ligation do I have to use for the PCR reaction???
I know it sounds like simple questions, but I need help!!!

in case of three fragments, i first ligate two of them using at least 300ng from the larger one(so i can see my ligation results on gel) i gel purify the ligated fragments and then do the ligation with the third fragment. it usually works. i generally use 1:3 v:i ratio. give it a try