Heat inactivation is not required for chemical transformation. And not recommended if you are using NEB quick ligation buffer

https://www.neb.com/faqs/2012/10/16/can-t4-dna-ligase-be-heat-inactivated

Also extend the ligation time to 1 hour.

You can avoid heat inactivation step.

First, you should check whether the ligase enzyme is expired or not and accordingly extend the ligation time.

You can also keep ligation mixture for different time-points like 1 hour and the other for 2 hours or accordingly.

Hi,

I would do the ligation and incubate at 20 degrees C for 3 hours following overnight incubation at 4 degrees. If the restriction sites are very close to each other, it sometimes gives hiccups.  And make sure there is no partial digestion. 

And one more thing, Heat the ligation mixture up at 45 C for 3 mins just before doing the transformation. It would have been convenient to help you if you mentioned the plasmid backbone name and name of Restriction Endonuclease site that you used.

@Mangal  @ Priya @Abir   Thanks for the reply. I was buying new T4 ligase and buffer; I will skip the heating  before the transformation and extend the ligation time. 

@Abir  I added the information of the plasmid and RE in the description. The restriction sites are very close. 

Hello,

did you also check the efficiency of both enzymes by cutting with Sgf1 and Mlu1 alone? if the efficiency of one or both enzymes isn't close to 100% you may end-up with a significant fraction of your destination vector cut only by one of the two enzymes (or no cut at all in the worst scenario). Even if this partially cut vector is a small fraction of the total, ligation is extremely efficient there so you may end-up with a large fraction of your colonies containing only the empty destination vector.

How many colonies did you get after transformation? if the number is high and you detect no insert at all you may consider:

- increasing the efficiency of the digestion (increasing incubation time and/or enzyme concentration or using a new RE vial).

- dephosphorylation of your target vector after digestion and before ligation al this will prevent self-circularization of partially cut destination vector.

Hello.  Are you sure about the nucleotide sequence of your "interested gene" in your tube?  If your sequence is downloaded from some databases and your gene is isolated by yourself, the sequence of your gene may be different from that in the database, according to the polymorphism.  In the case you use restriction enzymes A and B, for instance, for directional cloning,  Unexpected existence of the A site just close to the B site may cause a gene fragment (of almost the expected size) containing the cohesive ends created by Enzyme A on both sides.  

Since you get colonies, I can also only emphasize what was already mentioned and add some of our own experiences, maybe some of them may be helpful:

Quality / functionality of the ligation:

Actually the weakest spot often is the amount of ATP in the ligation buffer. We aliquot the buffer after arrival to prevent ATP degradation due to too many freeze/twaw cycles. But you can easily add extra ATP. In our hands we never had problems with the enzymes but actually with the ATP in the buffer.

DNA ratio:

Again I can only tell you from our experiences so by no way this suggestions are universal applikable. But based on the Insert size compared with the vector size, we have different protocols we used quite succesfully. For smaller inserts (roughly below 500 bp, this are for example new MCS, sh/miRNA epxression cassettes, localisation signals...) we work also with a 8-10:1 ratio (Insert:Vector). For longer fragments (from 1kb up) we go down to a 3:1 ratio or even a 1:1 (over 5kb). Keep in mind that the insert fragments can also be ligated to each other, so ratio has to be balanced with fragment size. If it is only one ligation, you could also test several ratios at the same time.

Total amount of DNA - Insert + vector:

We never go below 100 ng and never exceed 200 ng with preferred range 100-150 ng. Just tested with our own ligations and it gives consistent good results. 

Restriction enzymes:

We control every enzyme in a seperate digestion using the same conditions / buffer systems. It seems like a waste of time and material at first, but we actually rather saved time with it. Once we had one of these d*** automatically defrost freezers and our EcoRI was not working anymore (guess how long it tool us to find the problem ;)).

Also for the MluI - Do you use a high fidelity version? We used the normal version and it was never 100% working (independently of the company) so we made consecutive digestions and cut out the linear plamid... and switched to the high fidelity version later on and never had the problem again!

Dephorsphorylation of the digested vector:

I can only agree with Rocco Piazza, that this would also help with a more difficult ligation and/or with longer fragments. It also is an additional control to verify the success of the digestions. 

Time / temperature / PEG:

We don't do blunt end ligations anymore (it is actually easier for us to create a new MCS) since there always were higher mutation rates (single nucleotides missing at the ligation ends) than with any other method (overhang, directed as well as undirected; Gibson assembly and so on). So this applies for directed overhang ligations. We tested different temperatures (4°C, 18°C, RT, 37°C), times (5 mins up to 20h), addition of different PEGs... In the end we stayed with 5 to 15 mins at RT with no additives. With two different overhangs, fresh ATP, working T4 DNA ligase, a good ratio between insert/vector, total DNA amount... it works just fine. 

Good luck, I hope in between all suggestions you may find a solution.

@Claire ATP is indeed the weakest spot. I bought new ligase and ligase buffer. Problem solved! 

Thanks all! 

Glad to hear! :)