did you first check the purified DNA on agarose gel electrophoresis? after digestion.

And check some times ligation buffer & ligase enzymes does not work properly.

Some times transformation protocol may be a problem.

check competent cell viability is there or not? 

Hi Srikanth! Thank you for your answer. 

Yes, I checked the digestion on an agarose gel and I cut only my band of interest to proceed with GeneClean protocol. About ligation, I bought a new Ligase and I guess that it is correct. I also checked the ligation on agarose gel electrophoresis (Figure) before I transformed and I saw a ladder of bands, (I don't see the two original  fragments so I think ligation is working). 

I transformed bacterial strain with pcDNA3 (control plasmid) and the results were correct. 

In the figure, the first lane is 1Kb plus DNA ladder , the other two lanes are different ligations

Thank you. 

What's the concentration of Amp u are using? I hope your not heating the spreader for v long time maybe it's high temp. killing all the cells/bacteria. Also are you sure ur transformation protocol is working? why don't you try electroporation!!?

Please take more ligated product (5ul) for transformation as one is never sure about the efficiency of ligation 

Hi! Thanks but I have already tried that and it dind't work neither. 

Hi Macarena!

I can add to the suggestions given above.

1. Your insert is quite big, 8.7Kb and your vector being 10Kb. So there will not be much difference in the molar concentration (8 and 10 Kb). So, calculate the molar ratio correctly using online tools like NEBcalculator. Achieving successful ligation with so less difference in molar conc will be a bit difficult, but the efficiency can be increased.

2. You can set up some controls to check in which step exactly the problem lies.

For eg- a) use digested vector with ligase. If colonies appear then your vector might not be digested by both restriction enzyme. So, if RE1 has worked, the vector has sticky ends for RE1 which can be easily ligated and will give rise to colonies.

b) digested vector without ligase. If you get colonies your digestion is again incomplete. You can be sure that those many number of vector molecules are completely undigested. So, incomplete or no digestion will lead to no ligation again.

c) undigested vector transformation to check the transformation efficiency of your competent cells. As your ligated molecule will be quite big, you'll need high competency for successful transformation. If not, even if your ligation works, you won't get colonies, as we know the transformation efficiency is inversely proportional to the plasmid size, which in your case is 18Kb!

3. But if you are getting no colonies at all, then probably it is the efficiency of transformation that you want to improve, as at least some false positive colonies (due to vector re-ligation or undigested vector) should appear.

4. As already suggested increase the amount of DNA to be used for transformation. May be ligated molecules are less in the reaction mix and if you use even lesser DNA, you are reducing the chances of transformation.

5. Use higher volume for ligation. Your DNA molecules (both vector and insert) are quite big, so there will be molecular crowding. You should not have more than 10ng/ul DNA in your ligation mix. 

6. Use varied temperatures and conditions for ligation. I have used from 37 degree 10 mins to 16 degree 16 hours. Due to much higher kinetic energy and molecular motion chances of ligation is higher at 37 but many times it is random, At lower temperatures, the random motion is restricted allowing the sticky ends to form proper hydrogen bond contacts. So for each construct, any one of the conditions may be optimal. You need to optimize yours.

7. After ligation, take small amount and check in gel whether you see a upshift to check whether ligation has worked or not. 

8. As already suggested check your comp cells using them for normal smaller size plasmid transformation. Vary the conditions and maximize the efficiency.

Hope it helps!

Prepare fresh competent cells, check located DNA quality also, optimize competent cells and DNA ratio along with transformation temperature and time, use soc media for 1 hour incubation, use freshly prepared lb+amp plate, spread 100 ul in one plate and pellet down rest gently add 100 ul soc and spread in another plate ......best of luck

Hi Leon,

I also faced similar problem which was solved as the problem was in competent cells. Previously I used top10 cells which were causing problem. then I changed to DH5a which proved to be useful. You can also try to change competent cells. also do a control reaction using empty circular plasmid to check efficiency of competent cells. Also I suggest you to use Infusion HD enzyme or Clon express II one step cloning kit. They have better efficiency to ligate large size insert (in my experience). Also I wonder whether your ratio 1;3 is fine or not. Try different ratios of Vector to insert like 2:1 or 1:1. Please consider these things. . Good Luck