Have you disabled dephosphorylase before ligation, you can do it by heating or purifying your vector

I am not familiar with the pET 21 A, but if it is like the pcDNA3.1,May be the problem is not the ligation but the insert digestion ?

Yes Denis I have done heat inactivation at 75 10 mins followed by PCI and alcohol ppn as per Takara's instructions

Mohamed Insert digestion was done along with vector for 3 hours -see the gel picture here Lane 6 and 7 marked SDA is the insert both uncut and cut

I prefer purify the DNA with a kit type (Gel Extraction Kit, Q-Spin, omega biotech).

there is less inhibitor ligation.

to limit the action of the inhibitors, you can put half vector and insert and suddenly half of inhibitor.

If you feel that your ligation efficiency is poor, i think u you can add volume excluders like PEG in your reaction mixture to improve ligation efficiency.

your ligase can be dead. Take insert alone or vector alone and do o/n ligatino and then run on agarose gel, you should see a ladder, 1 insert, 2 inserts together, 3 etc.

Also, the most sensitive component of a ligation rxn is ATP. I always add it to ligation rnx, check with a bible re ATP conc (likely 1mM). Experienced molecular biologist always aliquots ligation buffer into many aliquots in order not to freeze-thaw ligation buffer too many times to keep ATP alive.

If you purified insert and/or vector from agarose gel there may be ligase inhibitors. If nothing else works, try to digest vector and insert and to ligate everything together without any prior purification or dephosphorylation. If possible cut the vector that contained the insert with an additional restriction enzyme to inhibit religation. By adding insert:vector at ~100:1 or so, the likelihood of getting the correct product should be reasonable. The suggestion of adding ATP is good. Adding DTT may also be a good preventive measure.

running ligation rxn on a gel will tell you if ligation worked (see above), you will see a ladder. If you are using TE to elute/store DNA after agarose purification, the EDTA from TE will chelate ions (Mg ions), so elute in warm 10mM nuclease-free TrisHCl. Storage of fragments with sticky ends will result in degradation, it is best if you ligate fragments the same day. If there are signs that some ligation takes place but u see no colonies, borrow electroporator, set it on max allowed setting, and electroporate, on average u get 100x higher efficiency compared to CaCl-competent cells. There's reason why molar ration 3:1 is recommended, you may go higher like 10:1 insert/vector, but one will be getting concatemers of insert and this will inhibit ligation (assuming your ligase works)

It will be difficult to distinguish digested and undigested insert since the difference will be very small. I used to clone the insert in TOPO-TA 2.1 and then digest the product from the vector. In this way, i was sure that the insert is digested and visible in gel. Pls try it if you have topo- TA 2.1 vector.

Other suggestions would be, vary conditions of ligation- try 4 degrees. I hope you are purifying the insert after digestion.. Spike the ligation mix with ATP (store buffer in aliquots. repeated freeze thaw destroys ATP).. Also as suggested earlier by others, increase the ratio of ligation mix, use appropriate volume of ligation..

Actually most possibilities has been covered nicely above. So I think now you have to find out at which step of the whole cloning procedure is the problem?

My opinion is with Igor, that it is possible your ligation reaction is dead; because you mentioned that you didn't get transformants in both Vector and Vector and insert, but usually you get some background colonies even after phosphatase treatment especially if you cut using one enzyme as in your case. I recommend you to make sure that your ligation is working by including extra tube that contains vector only digested with EcoRI without phosphatase treatment (without gel purification, just PCI and alcohol ppn), if you didn't get colonies for this tube most probably the problem is the ligation reaction because this is straight forward re-ligation.

Good Luck!

Try spiking your ligation buffer with some fresh ATP.

i mentioned this, 1mM final concentration. "Fresh" means the tube, which has not been thawed/frozen multiple times. Just as dNTPs, ATP goes bad gradually after multiple rounds of usage (freezing/thawing). while one always aliquots dNTPS, hardly anybody aliquots the ligation buffer, as one should. Well managed labs always have aliquots of ligation buffer specifically to avoid such mishaps with ligation

You could try another approach like Gibson cloning.

We have always aliquoted ligation buffer.

But the ligation doesn't work anyway :o))

it could be a bad lot of buffer, ATP died. Adding ATP from a reliable stock frequently helps. Going to next door lab and borrowing their ligation reagents (which they are happy with) will tell you if the problem is in the ligation rnx or in your DNA fragments/restriction enzyme. There may be traces of nuclease in restrictino enzyme or your water/buffer, etc.

Well thanks for all the answers

I am trying a self ligation with single cut Vector and after aliquoting my existing buffer which already has ATP ,I am also testing the same with Lamdba Hind III as a positive control

Dear friends

I tried self ligation with vector and it seems to be working so I am now optimising Vector plus Insert ligation using higher amount of enzyme and running the gel right now

Good luck for you!

Hey, I saw that you have still hard time with your cloning. If after all that didn t work may be (I hope no) you ordered wrong primer for you insert. That s mean wrong sequence for Ecor1 site (and usually I add TAAA in 5' before the restriction site).

Regards,

Thanks Mohamed-you are right -hard time a lot of attempts -my primer has the CCG in front of the ECORI site as recommended by the NEB site

In fact I am going to sequence the PCR product along with the cut insert to verify if it is OK