Dear Eric,

Kindly check the E.coli DH5a whether the bacteria is live or dead by plating them in agar plate. If they are alive,  try to transform the cells with some other known Plasmids / Undigested vector without insert. if again the problem continues, then your antibiotic resistant gene might be mutated. Check the concentration of Antibiotic in the agar plate. Try to increase the incubation time in soc media up to 1.30 hr. Increase the volume of soc media to 1 ml and re-suspend the pellet with 200 to 300 ul of soc medium before plating. you can increase vector : insert ratio up to 1:10. Try to add excess ligation mixture (20ul) to the DH5a cells. Finally keep the agar plate in the incubator around 16 to 20 hr as some time the colony will take time to grow.

Best wishes..

Dear Salazar,

It seems like your insert and vector preparations are fine. Ligation is sometimes tricky but since you checked for it, i think that is not the problem. Can you please set up some controls so that we can know where exactly is the problem.

1. DH5alpha is Nalidixic acid resistant, Streak them in a fresh plate and see how they are growing. Pick a single colony and make fresh competent cells if you can.

2. Take digested empty vector with ligase and use it for transforming these cells. If colonies appear then your digestion is not complete. But you can be sure that your ligase enzyme and reaction set up is working fine.

3. Take the digested vector and transform the cells without using ligase. So, if any plasmid molecule is left undigested that can enter the cells and you'll get colonies. 

4. You can use undigested vector to transform cells and check your transformation efficiency and competency of the DH5alpha. 

As a final suggestion, I think your construct size 7Kb+2.2Kb=9.2 Kb which is quite big, so you would need highly efficient cells for transformation. So, may be you should remake your competent cells. I feel the problem only lies with your transformation and efficiency of competent cells.

Hope it helps!

Hello Tias Saha and Perumal 

I already did some your suggestions, I prepared new batch of E. coli DN5alpha and check the efficienty, they are very good using a plasmid as big as the one I trying.

I will transform with the digested vector with and without ligase to see if I get some colonies.  

Thank you!!

I did all the controls suggested here. And I still have no colonies with my construction. Plasmid digested no ligase did not colonies. Plasmid digested with ligase no colonies. Undigested plasmid a lot of colonies.

I will try cloning smaller fragment to see if it is an effect of the insert. 

Hey Eric,

Have you checked the ligase eficiency? I always use T4 ligase that works for both sticky and blunt ends  (T7 ligase doesn't works for blunt ends ligation). If possible, you may try using different restriction sites for cloning. Also for increasing transformation efficiency, you can use competent cells prepared with Rubidim Chloride or use TOP10 competent cells.

Best Luck

Hello Eric:

The plasmid that you are trying to generate is big and maybe not stable, assuming that you did the basic trouble shooting, such as checking that the enzymes used in the cloning strategy are working well I would do as following:

1.     1:1 and 1:3 ligation ON at 16 C using Rapid DNA ligation kit.

2.     transform in Ecoli StabL-2 at 32C

Best of luck,

Marga

Thank you so much!!.

I´m still having troubles. I will try with shorter fragments.