Recent replies to "No ligation! Can't understand why!"

Reply from Sheldon Broedel, Jr.

Sheldon Broedel, Jr. — Tue, 12 Aug 2008 12:56:00 -0000

May I suggest an alternative approach. NheI has a 5’ overhang and SacI has a 3’ overhang. You should be able to explote this by annealing one strand of your synthetic oligo to the target vector (digested with NheI and SacI). Then use Klenow Fragement of DNA polymerase to synthesize the complement strand in place. Ligate and transform. There are specific protocols for this technique in several of the manuals on Molecular Biology Methods (e.g. Sambrook et al, 1989). My team has used this approach successfully for oligos of up to 120 bp. Good luck.

Reply from Ritu Chandna

Ritu Chandna — Mon, 11 Aug 2008 05:36:36 -0000

hello, i have also done ligation of 81bp duplex oligo into a double digested vector. ru sure that ur double stranded duplex oligo has the sticky ends complementary to the vector..i mean to say there might be strong secondary structures. for this use Oligo analyzer..it is a free software provided by IDT..try annealing the single strands again…i would also like to know what are the melting temps of the strands and the procedure u followed to dissolve the oligo and also the annealing of the single strands…hope u r successful in the cloning …

ritu chandna

Reply from Cinzia Pezzella

Cinzia Pezzella — Sat, 29 Mar 2008 10:33:49 -0000

Hello, I’m new in this forum discussion and today I wanted to post my problem..then I discovered It was the same problem you have: no ligation and I can’t understand why!
As you, I’ve tried different DH5a batches, gel purified vector and insert, and tried up to 1:5 ratio in ligase reaction, but I can’t get non colonies. I’m using T4 DNA ligase from Roche,aliquotating the buffer to preserve ATP. I do the ligase reaction on 100 ng of vector in a final volume of 10 ul. The I’ve tried to transform 1, 3, 6 ul of ligase reaction in 50ul DH5a, but I can’t get non colonies. I have a negative control (ligase reaction without insert), but I can’t get colonies too.
These are the details of my cloning:
I’ve cloned FUR gene in pGEMTeasy vector, then digested this vector with AgeI and ClaI (within the FUR gene) to subclone another insert (LEU2,digested with compatible ends NarI, XmaI) in the FUR gene.
Both vector and fragments have been gel purified after digestion (Leu2 fragment comes from a pUC19 vector containing Leu2).
- Do I have to inactivate Ligase before transforming?
Please answer me!!!!