Hi there,

There are three main issues in cloning: digest, ligation and transformation.

Do you perform single or double digest? If single then digest efficiency is easily spotted on gel by the linearization of the plasmid (run the gel long enough to possibly separe efficiently linearized plasmid from circular relaxed form which run close from the linear form). For double digest, it's more difficult : does the double digest results in the production of a fragment you can see on gel? If yes then this product will tell you everything is fine. If the sites are closer (ie fragment is shorter) it might be an issue because in double digest there might be a competition between enzymes for the sites and/or digest site near to DNA extremity issue. Sequential digest might be the solution provided you have checked both enzymes are efficient individually. The same issue of DNA end proximity arises if the insert you intend to clone is a PCR product (you have to include at least 3 extra bases beyond the digest site to optimize enzyme efficiency). Do you gel purify the digested insert too? This is advised to get rid of small bits generated by digest. Control DNA concentration with nanodrop after extraction/purification or on gel with calibrated DNA ladder to estimate the amount per band.

For ligation : check ligase activity with a self ligation assay with a plasmid linearized by a single sit digestion. Run 2 controls with your specific vector : vector alone (to evaluate the presence of circular plasmid) and vector alone + ligase (to evaluate the presence of linearized plasmid by single digest). In the ligation mix, use 100ng vector maxi and 3:1 to 5:1 insert:vector molar ratio (if vector is 5kb and insert 1 kb, you will use 60ng to 100ng of insert for 100ng of vector). For incubation, the longer is the better (overnight at cool temperature is the best).

For transformation : check competency at the same time as you run the actual transformations with 0.1ng of pUC plasmid for instance (this control should give you at least 100 clones as 106 cfu/µg of DNA is the threshold for competency in cloning experiment. If chemical competent cells, the longer the better for incubation on ice once cells and ligation are mixed. Allow the cells to recover in rich medium without antibiotics for at least 30minutes before plating (and even longer if possible). Plate the same amount of cells on different plates in order to be able to compare the number of clones obtained.

Good luck

After the ligation reaction you can check if the ligase was able to ligate your DNA fragments. Here you can find information on how this is done:

http://www.thermoscientificbio.com/whats-new-t4-dna-ligase/

If this experiment shows you that you have a positive ligation reaction then you can rule out the possibility that your ligase failed. It might at least help you coming closer to solving your problem...

Good luck!

Several controls needed to optimally trouble shoot your problem(s). After gel purification have you checked to confirm you have DNA and in the correct amounts? Are you sure you are using LB plates with the appropriate selectable marker (Amp/Kan) and that whoever made the plate labeled it correctly? There is almost always some background colonies from the purified vector so if you have none it suggests maybe you are not using enough DNA in the ligation or transformation. Is uncut plasmid your positive control? Are you using an optimal transformation protocol? We use both quick (1 hr) protocols and can get quite different results, though the quick protocol is not recommended when Kan involved.

as suggested by others, there could be several problems, right from the concentration of DNA after gel extraction to its purity. U need to ensure that the DNA of proper purity and concentration is used for ligation. Also u need to have a linearized plasmid as a positive control to ensure that the ligase works fine; and an uncut plasmid to ensure that the transformation was done properly.

Hi there,

There are three main issues in cloning: digest, ligation and transformation.

Do you perform single or double digest? If single then digest efficiency is easily spotted on gel by the linearization of the plasmid (run the gel long enough to possibly separe efficiently linearized plasmid from circular relaxed form which run close from the linear form). For double digest, it's more difficult : does the double digest results in the production of a fragment you can see on gel? If yes then this product will tell you everything is fine. If the sites are closer (ie fragment is shorter) it might be an issue because in double digest there might be a competition between enzymes for the sites and/or digest site near to DNA extremity issue. Sequential digest might be the solution provided you have checked both enzymes are efficient individually. The same issue of DNA end proximity arises if the insert you intend to clone is a PCR product (you have to include at least 3 extra bases beyond the digest site to optimize enzyme efficiency). Do you gel purify the digested insert too? This is advised to get rid of small bits generated by digest. Control DNA concentration with nanodrop after extraction/purification or on gel with calibrated DNA ladder to estimate the amount per band.

For ligation : check ligase activity with a self ligation assay with a plasmid linearized by a single sit digestion. Run 2 controls with your specific vector : vector alone (to evaluate the presence of circular plasmid) and vector alone + ligase (to evaluate the presence of linearized plasmid by single digest). In the ligation mix, use 100ng vector maxi and 3:1 to 5:1 insert:vector molar ratio (if vector is 5kb and insert 1 kb, you will use 60ng to 100ng of insert for 100ng of vector). For incubation, the longer is the better (overnight at cool temperature is the best).

For transformation : check competency at the same time as you run the actual transformations with 0.1ng of pUC plasmid for instance (this control should give you at least 100 clones as 106 cfu/µg of DNA is the threshold for competency in cloning experiment. If chemical competent cells, the longer the better for incubation on ice once cells and ligation are mixed. Allow the cells to recover in rich medium without antibiotics for at least 30minutes before plating (and even longer if possible). Plate the same amount of cells on different plates in order to be able to compare the number of clones obtained.

Good luck

There are many factors that could be bothering the ligation

-Are the ends compatible?!

-Column elution made with water or buffer (sometimes water si better but check pH)

-Sizes of vector and insert. usually 1:3 and 1:5 (ng) works fine with me being 1=50ng

-Blunt or cohesive ends?!

-Usually an overnight ligation at 16°C works fine

-For difficult ligations you could phosphorilate the insert (and purify again before ligation)

-Good concentration and good DNA quality is crutial

Good luck

Dear Gaurang,

After some excellent suggestions, I would just like to add few things-

1. If your positive control is working, then your competent cells are fine and so as the procedure of transformation you use. 

2. Check the ligase enzyme efficiency by setting up controls like-

a. single digested vector without ligase

b. single digested vector with ligase.

The number of colonies will tell you  the efficiencies of your single digestion and ligase.

3. Re-check your ligation conditions. Cohesive ends anneal best at lower temperatures like 4 degree. You can incubate at 4 degree for 24 hours. Also modify the vector to insert ratio to 1:5 or may be 1:7. You may use the NEB calculator to find out the molar ratios. You can set up with both the ratios to see which one works.  Also try to use more volume of your ligation mix to transform the competent cells. Heat denature the enzyme before you use it for transformation. As already suggested, you can also check the ligated molecule on agarose gel. I'm attaching two links which will help you more with these concepts.

4. Lastly, you can try to use a different vector backbone with a different selectable marker and see if that works. Some times, some leaky protein expression produces toxic proteins which might inhibit the cells to grow. 

http://nebiocalculator.neb.com/#!/ligation

http://bitesizebio.com/10203/ligation-optimization/

https://www.researchgate.net/post/What_is_the_best_temperature_and_incubation_time_for_successful_ligation

Give us more details about your protocol.

-Check you buffer. Ligase requires ATP. Repeat freeze/thaws will degrade the ATP, resulting in no reaction. It's best to aliquot the buffer that comes with the enzyme so you're not freeze/thawing the whole batch every time.

-If your buffer contains PEG, stay at room temperature, don't heat or freeze or leave the reaction for long/overnight incubations because it will decrease the transformation efficiency. Without PEG, your reaction may need to be incubated longer. The T4 ligase protocol for buffer without PEG says room temperature is fine, but in my experience it is better to use 16C overnight and sometimes as low as 4C. The limiting factor usually isn't the enzyme activity, but rather the DNA coming together in the right configuration for ligation. If you're using sticky ends, note that lower temperatures makes it more likely the ends will transiently bind together.

-If your ligase enzyme is getting old, try adding 1 uL per 10 uL reaction, instead of 0.5 uL. In my experience, the buffer usually goes bad before the enzyme, but ask your labmates if they've been having trouble with ligations. If so, and the ligase is expired a few years, you might need a fresh stock.

-Speaking of enough enzyme: check your P2 pipette. If you're trying to add 0.5 uL with a poorly calibrated pipette, you might not be adding all or any of it. Try adding 1 uL or 2 uL ligase instead.

-Is your insert phosphorylated and in dsDNA form? If you're using insert straight from regular PCR without any enzyme digestion, or you made insert by annealing oligos, you need to add a 5' phosphate to your insert. I use T4 PNK and then heat inactivate. I don't purify it out, but with short DNA segments (8 kb plasmids). For plant vectors (often >12kb), I try for 60 ng minimum.

-Your cells might not be competent enough for ligation reactions (which don't make much circular product). Ask your labmates if they are also having issues with cloning. Add

Mr Dominique Liger, i use double digest restriction fastdigest saci and hindiii. I got many colonies but no one of them have recombinant plasmid that i want. After i analysis, i guess self ligation. Can this happen?