Make sure that you have carefully applied  the protocol that allows you to make TOP1 0 and DH5 cells competent if you are certain that the ligation worked or maybe too much ligation mixture was used (use < 5µl of the ligation reaction for the transformation).

Look at the link below it can help you

https://www.neb.com/tools-and-resources/troubleshooting-guides/troubleshooting-transformation-reactions

Thank you so much.. One more thing. I ran another ligation gel and i m getting extra band in between my insert and vector in my Ligase negative sample. What i mean is A sample without Ligase has Insert (1kb) and vector (6kb). I ran the gel and i m getting another band in between the two. I then ran gel from purified digested product and no band there.

An extra band might be due to some contamination.I would suggest you to go for a fresh digestion of products followed by transformation.Generally I take a ratio of 1:3-1:5 for ligation.you can also try the same.

Are you using agarose gel electrophoresis to purify your PCR products? If so, be aware that DNA damage from UV irradiation produces exactly those symptoms, i.e. DNA that ligates fine but does not transform.

If you are directionally cloning, self ligating inserts or vector should show no gel multimers and no transformants.  Ligation of insert and vector should show a discreet product by gel and colonies after transformation.  Cut a "sister" lane for vector and insert and examine by UV. "Patch" the gel together and cut the unexposed lanes for purification.  Perform a ligation control with lambda/HinDIII ladder.  If you are using commercially produced cells, use the control vector and dilute your ligation to 10 picograms/ml.  Check your water baths with quality thermometers.  Shout out when this works!

Is there a possibility that the restriction enzyme you used cut the selection marker? Also, what is the rough yield of digested products after gel purification? The 3:1 ratio works well for me too and typically, I use about 30 ng of backbone with 90 ng of inserts. Perhaps try a different ligation brand. We use T4 ligase from NEB. Good luck! 

Check the efficiency of your competent cells. Lack of proper storage may lead into lack of its efficiency.  

Also, try setting up the ligation reaction from fresh PCR products, becoz the A tail in the PCR product is stable only upto 24 hours...

As there are 'n' number of variables, you have to take special care of the storage of your reagents, vectors and PCR products. Also check the buffers if it is contaminated.

If you are SURE that it is the transformation that is not working: Use a small volume of the ligation reaction to transform your competent cells, at most 10% of the volume of the competent cells (5uL for 50uL of cells at most). Incubate for a longer time (at least 1 hour) with SOC media and no antibiotic (as opposed to LB) after heat shock to allow expression of antibiotic resistance gene, and plate the entire volume.  

However, I would go back and ensure that there are no other steps that could be affecting the transformation, such as, deactivating the ligase prior to transformation may inhibit transformation if the ligase buffer contains PEG. The presence of ethanol, and other impurities can also inhibit transformation. Also use various molar ratios of vector:Insert during ligation, etc. 

I was having troubles with my ligations/transformation for awhile as well, for me it turned out to be two things: Not using the optimal molar ratio of DNA:Insert  and later on I used the wrong restriction enzymes because I made a silly assumption about my vector. Thankfully though, things are working now. 

Thanks everyone. Turns out the problem is ligation and not transformation. Uncut is giving me colonies. My assumption 1 is ligation buffer so i have changed it and now trying it. Also my assumption 2 is the molar ratio. One of my friends suggested 50 ng vector and 150 ng of insert. I might try it out. My assumption 3 is ligation on the whole so trying EcoRI cut on pUC19 and see it ligation works there. Fingers crossed.

Hi Naushad... not sure what you are up to now with your cloning, anyway just as a general rule, digestion and ligation are in the vast majority of cases the critical steps in cloning.

While vector digestion (unless you choose two RE too close to each other... watch out for that!) or insert digestion from a previous vector is normally relatively simple and flawless, digesting efficiently a PCR fragment can be difficult or impossible, for  biological reasons: the restriction site is terminal on the DNA sequence and on one side of it there are no or (if you have been thoughtful designing the primers) few nucleotides for the RE to sit on. REs react very differently to cutting on a 5' or 3' end of a fragment. In general, designing primers with 5 nt extra to the end side of the restriction sequence helps, but there is no guarantee, which means that often digestion of a PCR fragment is inefficient... and you have no means to verify it!

Consequently, it is suggested to use 3 to 5 times the molar ratio of insert compared to the vector. Molar ratio...! So, using 50 ng of vector and 150 ng of insert per se makes no sense... the ratio will depend on the size of the vector and the insert. So, the ratio described here is good if your insert has the same molecular mass as your vector (for example they are both 4 kb long). If your insert is 1/10th of your vector (say 400 bp), the ratio you are using is way too high, you are putting together 30 molecules of insert for each molecule of vector. In these conditions the ligase will just join inserts in a head-tail long chain, because of easier statistical encounter. That's the smear you see on your gel.

Finally, ligation kinetics have their rules! In order to favour vector-insert encounter the volume needs to be small. So ligation is performed in 10-15 microl. maximum. Additionally, if your REs leave sticky ends, you need to favour their annealing, which means ligation will be most efficient at 15 °C (O/N, since the enzymatic efficiency will be reduced compared to the normal working temperature of a ligase, that is 37 °C). If the ligation is blunt ended, you'd better favour the ligase efficiency so that it ligates fragments quickly as soon as they meet, so 20 °C O/N will be optimal (37°C are seldom optimal due to too much kinetic energy of the DNA fragments and convective movement of the solution, all making it very difficult a three component encounter - vector, insert, and ligase...). If you have one sticky and one blunt end RE, you can ligate them one after the other, O/N at 4 °C and when you arrive in the lab in the morning shift to 20 °C until evening, when you can transform.

Good luck.

So a little update now. I thought my ligation wasn't working so I brought pUC19 as my control. I digested it with EcoRI and then ligated it again (+) and (-) ligase. I got good colonies from (+) ligase and very very few colonies from (-) ligase. This experiment suggests that my ligation is now working. 

The only thing that I didn't try here from Pietro's and other's protocol is overnight ligation. I used 1 hour at room temperature. I guess, I will use O/N for my insert (1kb) and vector (5kb) ligation experiments. Lets hope it works this time.

Also be sure to kill the RE.

So turned out ligation kit was the problem. I repeated everything and got few colonies. As a confirmation i rerun restriction digest with similar enzymes. I got the products too but i am getting another band in between the two bands that is present in uncut sample too. Im not sure what it is.

which protocol did you use for providing competence cell. although electroporation is more sensitive, it has a lot of challenge. I think it's better to use heat shock.

another thing, did you check the efficiency of competence cell?

Hello. I have some experiences to share to you.

- Your competent cells should be usable - during 6 months for heat shock method, and during 1 month for electroporation. 

- You should try heat shock. It is easy to carry on. 

- Your DNA need to be high enough for transformation.

- Ligation ratio (normally I use vector:insert 1:3 or 1:4)

- Ligation at room temperature for sticky end, and overnight at 14 degree for both sticky end and blunt end

- During transformation, recovery step is very important, so you should check your protocol.

Next is your cutting result. I think there are some possibilities:

- Your plasmid is contaminated by other DNA

- Or I think your cutting is not perfectly (1 band of uncut, 1 band of 1 cut, 1 band of 2 cuts). And uncut plasmid might have 1-3 bands because of its structure (fold or unfold). 

Good luck !