Many things can go wrong even with cloning experiments that seem easy. I would first eliminate the AP step. If you are doing a directional (double-digested) cloning there's no need for the AP treatment. Furthermore, AP is tricky to eliminate and any remaining activity will actually dephosphorylate the insert as well...

Second, the ligation reaction. In a total volume of 10 ul (vector+insert+buffer+enzyme+H2O), ligate overnight at 12-16C gradient (add some ice to water up to 12C and incubate overnight).

Third, beware of the ligation buffer. T4 DNA ligase requires ATP, and if the buffer is very old, have been thawed many times, or conserved at temperatures other that -20C it is possible that the ATP is gone.

And last, but not the least, use the highest efficiency competent cells commerciallly available. I use JM109 at greater than 10e8 cfu/μg and belive me, !that mades the difference!

Hope this helps

estanis

Many things can go wrong even with cloning experiments that seem easy. I would first eliminate the AP step. If you are doing a directional (double-digested) cloning there's no need for the AP treatment. Furthermore, AP is tricky to eliminate and any remaining activity will actually dephosphorylate the insert as well...

Second, the ligation reaction. In a total volume of 10 ul (vector+insert+buffer+enzyme+H2O), ligate overnight at 12-16C gradient (add some ice to water up to 12C and incubate overnight).

Third, beware of the ligation buffer. T4 DNA ligase requires ATP, and if the buffer is very old, have been thawed many times, or conserved at temperatures other that -20C it is possible that the ATP is gone.

And last, but not the least, use the highest efficiency competent cells commerciallly available. I use JM109 at greater than 10e8 cfu/μg and belive me, !that mades the difference!

Hope this helps

estanis

Hello, I faced similar problem but in petduet1 vector.Initially when I tried with 3:1(insert :vector) ratio and ligation 37 degree C for 2 hours, It didn't work for me.Then I tried with same ratio but different ligation conditions, 16 degree C for 16 hours, and It works fine. Further during transformation use less than 10% ligation mixture (10 µl mix in 100 µl comp cells), the salt and other compounds present in ligation mix inhibit the transformation.

Good Luck.

I agree with the comments above - 16 degree overnight ligation for sure, plus make sure the ligase buffer is fresh enough as Estanis suggests re: ATP. I always use Ultracompetent XL1-Blue cells myself (from Stratagene) - either way the extra efficiency is worth the expense as you'll never get 10E8 by making your own competent cells.

Thanks a bunch, guys!

Best wishes,

Andreia

As Estanis says, dephosphorylation is not required when non-compatible cohesive ends are used. Besides, AP could have exonuclease activity.

16C overnight ligation may be a good alternative. You should also try with 5:1 ratio, it sometimes works.

I recommend you to protect the vial from light because ATP is light sensitive.

Hello Andreia

I had a similar problem in cloning few months ago,

You can increase ligation time to 14-16h at 16C (best ligation occurs at 16C ) and test the 1:5, 1:6 molar ratio of vector- Insert (with 60-100ng vector). Also, check the T4 ligase activity, this enzyme require buffer containing ATp. The ATP can be degraded by freeze-thaw.

In the end, I got the answer with completely pure insert and vector in ligation reaction.

Good luck

I second the recommendations concerning ATP in the ligation buffer. In my experience, home-made ligase buffers prepared with fresh ATP solutions will consistently outperform commercially available alternatives, by a considerable margin.

Also, how did you purify your band? Extensive irradiation on top of UV boxes while cutting out gel bands will usually ruin your cloning. See e.g. Hartman, P. S. 1991. Transillumination can profoundly reduce transformation frequencies. BioTechniques 11:747-748

Another source of trouble is incomplete digestion by either enzyme. Set up parallel single-enzyme reactions under the same conditions used for the double digestion to verify that the they are proceeding to completion.

Hi Andreia, it looks like you have a lot of good suggestions here regarding the ligation so I won't muddy the waters there. What I would suggest is in addition to making sure you extend your ligation time and try various temperatures ie. 16 over night or room temp 2 hours are the following.

1. Make sure your insert and plasmid are extra clean. Often a PCR purification after your digestion can make a world of difference in cleaning up your sample prior to ligation.

2. Check your transformation method. Fresh competent cells provide a huge push in terms of increasing transformation efficiency and if your ligation is tricky creating a fresh batch may help aid you in your goal. I would suggest the Hannahan method using rubidium chloride (http://vosshall.rockefeller.edu/protocols/Hanahan.pdf) to generate and then transform your cells. It has always given me great results and you can use a large excess of your ligation reaction with few ill effects.

3. Finally I would double check your plates and your plasmid/cell selective markers. It's often a silly but fatal mistake in that the wrong plates/antibiotics/selective marker are used accidentally or because of poor labelling.

4. Finally although the ratio of vector to insert is important I would error on the side of far too much insert to vector. That and if in doubt try adding a little bit of DTT to the ligation buffer if it isn't already included. DTT helps to sequester the DNA together increasing the changes of a successful ligation

Good luck and hope that helps!

Thank you so much, everybody! Found some precious tips around here, I really appreciate your help. Will let you know how things go around here...

Wishing you all a great weekend,

Andreia

When you write your ligation is not working, does this mean you do not have any colonies? Or that you only get religated plasmid?

There are several step you can start to chek:

- are you 100% sure of your sequences and restriction site presence?

- your products are correctly digested and purified? I alway prefer to "open" a plamid that will liberate a fragment to be sure both enzymes worked. For me, this point was the most important. Now that I have one plasmid well digested, everything goes in easily. I also male short digestions: 1h30 is enough. My favorite purification is with low melting agarose gel, phenol/chlorophorm extraction but not the column.

- Did you quantified your products with absorbance or in a gel using ladder such as Pst I Lambda? For your ratio if it is really not working, you can also try several ligations at time: 1:5, 1:3, 1:1 etc.

- For routine ligation such the one you are talking about I ligate this way:

1) Insert + plasmid + water - 65ºC 2min to "separate" well these fragments.

2) 2min on ice

3) add buffer and T4 ligase on ice. Incubate o/n 4ºC

- Last, did you check the efficiency of transformation of your competent cells? With 1ng of pBSK for example?

I had a lot of problems with ligation but with method, not quick and dirty, it use to work very well. Good luck with ligation

When you write your ligation is not working, does this mean you do not have any colonies? Or that you only get religated plasmid?

There are several step you can start to chek:

- are you 100% sure of your sequences and restriction site presence?

- your products are correctly digested and purified? I alway prefer to "open" a plamid that will liberate a fragment to be sure both enzymes worked. For me, this last point is the most important. Now that I have one plasmid well digested, everything goes in easily. I also male short digestions: 1h30 is enough. My favorite purification is with low melting agarose gel, phenol/chlorophorm extraction but not the column.

- Did you quantified your products with absorbance or in a gel using ladder such as Pst I Lambda? For your ratio if it is really not working, you can also try several ligations at time: 1:5, 1:3, 1:1 etc.

- For routine ligation such the one you are talking about I ligate this way:

1) Insert + plasmid + water - 65ºC 2min to "separate" well these fragments.

2) 2min on ice

3) add buffer and T4 ligase on ice. Incubate o/n 4ºC

- Last, did you check the efficiency of transformation of your competent cells? With 1ng of pBSK for example?

I had a lot of problems with ligation but with method, not quick and dirty, it use to work very well. Good luck with ligation

Hi Andreia,

At which temperature did you perform your ligation reaction? Based on my experience, I think overnight ligation at 16 C with 1:5 vector to insert ratio gives the best results. Also, as highlighted by Navarro, there is no need for you to incorporate AP in you reaction mixture.

One useful trick that has often helped me with cloning when using two different restriction enzyme is by employing an additional restriction enzyme (any site between the two REs you are using and that restriction sites should not be present anywhere else in your vector or within your gene ) in the LIGATION mixture. The purpose of incorporating the third restriction enzyme in your ligation mixture is to cut the religated plasmids and not the ones with your insert, so that you will only get colonies integrated with your gene of interest.

Good Luck

Hi Saranpal,

Thank you so much for the tip, it sounds great! One question, though: if I cut my vector with 2 REs, wouldn't I remove any restriction sites between them? How would the third RE work, then?

Thanks again!

Best,

Andreia

Hi again Andreia,

The addition of the third restriction endonuclease is just to eliminate self-ligated plasmids that may be present in your ligation mixture. The self-ligated plasmid will still contain other restriction sites in between the two RE that you have chosen. I normally omit the addition of AP for the same reason explained by Navarro and others, instead replace it with the addition of a third restriction enzyme, just before plating onto my agar plates. It works like a charm!!!

Hi, everybody!

I finally got it, thank you all for your help!

I believe that omitting the AP step was key. I have also modified the ligation conditions (new buffer with fresh ATP, 16ºC, overnight). Today I confirmed my 3 clones by RE digestion and am very happy :D Now, I'm sending them to sequencing to confirm.

Thank you all very much!

Have a great weekend,

Andreia

PS: Saranpal, I'm definitely incorporating the third RE on my next cloning reation ;) Thank you!

It's been a pleasure, and I hope that the clones are true

Have a nice weekend

Estanis