There can be several factors. It is slightly difficult to ligate 2kb insert. Did you quantify your insert and plasmid before ligation? what were the mol ratios between insert and plasmid? 

Sometimes you are just out of luck. I tried to clone a 2.5 kb insert into a 7 kb vector by simple PCR / digest / ligate for 3 months straight. There was no reason why it shouldn't work. I had cloned 2 kb into 8 kb for several already with ease. In the end I sort of gave up and switched to Gibson Assembly (using NEBuilder HiFi), which was kind of overkill. Got around 30-40 colonies, picked 8, all positive. So sometimes it may be better just to switch strategies, even if there is no real reason why. Benchling is a great tool to design Gibson assembly primer.

If you want to continue trying the classic way you may consider following factors:

- ratio of insert:vector => we normally use 3:1 but with your sizes I would go with 1:1

- do you get a lot of empty clones? Try overnight digestion of insert and vector

- do you get no colonies at all? Try overnight ligation on bench and maybe commercial ultra competent cells (I like NEB-10 or NEB stable)

- have your E coli S17 worked in similar conditions before?

Dear Rohora.

With a kind respect to all concpts. But I think your vector is very small and your insert is so huge. How did you provide your competent cells? This is a critical step.

There should not be any problem with that size insert and plasmid, it is not all that large. I would be sure to run all the appropriate controls. Frequently the competent cells people use in cloning experiments are not as competent as might be required for a cloning, so be sure to do an uncut plasmid control and you should get nearly a lawn of colonies upon selective transformation. Also make sure your ligase and ligase buffer are good. 

Dear Prof. Benedik.

The insert is not so large itself. I meant the insert size ratio to the vector is so huge. However, competent  cells preparation is a critical step.

Dear Nohora,

I agree with the comments given above. In addition, did you purify your ligation products prior to transformation? Did you use ethanol precipitation to purify your ligation products? Did you air-dry your DNA pellet to remove residual ethanol before you resuspended your DNA? Did you quantify your purified DNA using Nanodrop? What about the level of purity? Did you check its 260/280 ratio? Did you run agarose gel for your ligation products? You can use appropriate controls, such as cut vector, cut insert, uncut vector, for comparison.

Which method did you use for transformation? heat-shock? electroporation? or others?

You can see it from different perspectives to fix your problems.

Thank you all,  all of your advices are taken into account. I am reviewing the controls once more, the DNa quantification and the purity. I purified from gel the plasmid and the insert but i'll double check everything. 

But what was exactly your problem? Were you getting no colonies after transformation or the colonies were obtained but they contain the empty vector without insert? The ways to solve both problems are different. 

In the first case, higher effciency competent cells or even better electroporation can help to get enough colonies for screening.

If you get colonies, but the insert is not there, transformation is not the problem. To improve ligation you can try different molar insert/vector ratios (from 1/1 to 16/1). If your difgested vector and insert are not able to ligate in any condition, I would try to improve their preparation by

a. Using more enzyme and longer digestion times.

b. Doing separate digestions with both enzymes, changing the order to see whether one of them is not working properly. This can only be seen with the vector.

c. Purifying the digested vector from a gel after each digestion.

d. Dephosphorylating the vector after the second digestion and before purification.

1. Go for overnight digestion of vector and insert.

2. Ensure that no traces of ethanol during your elution from column.

3. Do keep vector control.

Please check condition for competent cells and you can elongate heat-shock time. (Normally, heat-shock time is 45s. You can increase it to 1 min or 1,5 min)

One more possibility. XbaI is sensitive to dam methylation. Make sure, that XbaI recognition site in your plasmid is not affected (or grow it in dam- strain).

Martin

Hi,

I would still recommend to go for the digestion from PCR2.1 plasmid.... then you know for sure your insert is cut. Go for large amounts eg multiple 5-10µg digestions and pool before column purification(s).

You need to have reasonable yield otherwise the concentraion measurement is not reliable. Check after insert purification again on gel... try to dubbel check the concentration of the insert using you DNA maker.

I usually go for multiple INSERT:Vector ratio's  (eg. 1:3, 1:1, 1:5)

Use fresh T4 ligase.... overnight incubation.... (no rapid kit)

Use commercial competent cells with a high cfu,

Good luck :-)

Remco.

Hello,

Just a small addition, that is useful sometimes: you could test your ligation by PCR (e.g. with primers on the backbone, or primers around the ligation sites) before doing any transformation. If you do get a product by PCR the problem is the transformation. Otherwise, the problem is at or before the ligation.

Also, I would say that if the cloning becomes too difficult (I mean if you do reasonable troubleshooting and it does not work, and you keep on trying and trying) you should consider the cost of your own time. I.e. if it takes you a month to get it to work, you will have wasted time and resources which you could have used to buy a Gibson assembly kit.

If a Gibson kit looks too expensive, you could just design a Gibson assembly, buy some T5 exonuclease to get 3' protruding ends, use some proofreading polymerase (Phusion, Q5, etc) you have in the lab, perhaps column-purify and then ligate with standard T4 ligase at 4C overnight, mimicking a cheaper, home-made Gibson assembly approach. You can see how the original Gibson works here (http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html?foxtrotcallback=true); note that they use Taq ligase instead, because it's more thermostable, but I'm suggesting to use T4 at low temperature to keep costs down.

Good luck!

Ruben

Dear all, I am so  greatful for all of your sugestion. As for the moment I have taken into account every thing here and currently doing one more round of Clonning and transformation. If it doesn´t work this time, I am definetly considering the Gibson assembly.

Thank you all, 

Best Regards, 

Nohora