Zhila,

You could make 3 digestion tubes to check if the enzymes are working correctly because when you double digest in a MCS within a plasmid and run the digestion in an agarose gel you are not able to see the fragment that is cut. In one tube you make the dobule digestion EcoRI/HindIII, in another you digest only with EcoRI and in the other only with HindIII (the 3 of them with the same conditions), and then run the 3 digstions in an 0.8-1% agarose gel to be shure the enzymes did work fine (you should see three linearized plasmid backbones)

About the ligation, a molar ratio from 5-10 is usually used to ligate blunt ends and in this case you are ligating cohesive and non-compatible ends and an insert:vector ratio from 1-4 should work fine. 

Maybe you could make 4 ligation tubes with different insert:vector ratios from 1 to 4.

Another ligation trick: once you finish mixing all the ligation reagents (included DNA), put the tubes in a "water floating rack" and put the rack in any water container (with water at 25 ºC) and put all that in the 4 ºC refrigerator for 1-2 days.

Good luck! 

Martin

I think you should check the ligase first, is it perfectly working or not.

And then the ligation time you mention is too short and too long. Did you try over night or 24h. and you should run 1% agarose gel. 

One more thing plz again check the size of your vector and insert. some times if the if insert is too long and vector is too short its ligate its viseversa

Good luck

Excellent suggestions already! But, I would like to add few points to it. As you have observed your ligation ain't working completely, there may be 2 reasons to it.

1. There is something wrong with your ligation mix.

a. For that, try and use fresh buffer which is not frequently frozen and thawed. As T4 DNA ligase buffer is likely to contain ATP, which might destabilize due to multiple freeze-thaw process.

b. You have already tried two different ligation conditions, so you might as well try a third one, which is 16 degrees and 16 hours incubation in water bath. 

2. Your vector and/or  insert is not completely digested to yield compatible ends for successful ligation. 

a. For that, you need to try both single digestions and double digestion as suggested. 

b. Increase your incubation time for restriction digestion (you can go upto 16 hours, check whether your enzymes are extended activity enzymes or not)

c. Also, a better way to make sure complete digestion of your vector, is to digest some other clone (different insert but same vector), digest it to release the insert, and carefully cut out the linearized vector from gel and purify to use it. That minimizes any chances of few molecules of vector remaining undigested or partially digested.

d. Also to make sure, your empty vector is completely digested, use the double digested vector without ligation (linear vector) for transformation and see if you get any colonies. Colonies will suggest improper digestion again. 

3. To check whether your transformation is fine, use empty vector ligate it using same conditions as your vector insert, and transform it to see if you get colonies. That'll rule out if there is any problem in transformation. As, your vector is quite big so your construct would also be big and hence might have lower transformation efficiency, so use good quality competent cells.

All the best! Hope it helps! 

Thanks all. I will follow up on your suggestions

Hi Zhila

The following works may be usefull:

1- Digestion separately and confirmation the digestion (transformation only by the vector so if you had  colonies, it means that the dig reaction was not complete and ...

2- Using serial ratio of insert:vector in ligation

3- check the competent cell by transformation of any ready to transfect vector