Nature Protocols Discussion Forum: topic
This is a public forum
No ligation!!!!!!!
Cinzia Pezzella
Saturday, 29 March 2008 10:38 UTC
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!!!!
-
Replies
-
Hi Cinzia,
in my experience, you do not have to inactivate the ligase – I hardly ever do and my ligations usually work. Reading your post, I see several possibilities:
1. Your ligase does not work properly. Do you have a neighbor/colleague you can ask for a few microliters, or do you have another aliquot?
2. One of your enzymes does not cut properly – even though you see the excised fragment on a gel, it does not necessarily mean that the generated overhangs are actually intact.
3. Try not to clean the fragments too often. In my experience, the more you do with the DNA (gel excision, column purification etc), the more likely you are to lose your sticky ends. I would try to not clean at least one of your fragments (vector?) by gel extraction, but instead to use a “quick-and-dirty” ligation procedure.
I hope this helps, good luck! -
Hi Cinzia,
Do you keep positive controls for ligation and transformation?
-