Dear Nhan,

First of all, thank you for providing with details of your experiment. I have made several constructs employing the strategy described by you. However, I do it little differently and it always works. Please try it like this:

PCR amplify the two fragments and purify them. Restrict the two fragments with XbaI and purify. Ligate the two fragments (50 ng each) in 1:1 ratio at room temperature for 60 min or at 16C for overnight.

Setup a PCR reaction; use forward primer (with EcoRI RE site) that hybridizes with fragment 1 and reverse primer (with BamHI RE site) that hybridizes with fragment 2. Use 1-2 uL of ligation mix as template. You should be able to PCR amplify the ligated product. Purify the amplicon, restrict with EcoRI and BamHI and purify again. Use this fragment to clone into the vector at the same sites.

Alternatively, ligate 100-500 ng of XbaI-restricted fragments 1and 2, purify the ligated product from the gel, restrict with EcoRI/BamHI, and clone in the vector at the same site.

Good luck.

hi, i've tried to do that and it is very difficult to get ligation fragments, my advise is to explore employ gybson cloning, it was the solution for me and very efticient.

You used a 60:1 ratio of insert:vector for cloning?

The protocol did not seem very clear at this point. A ratio of 3:1 insert:vector should be used. 

A ratio of 60:1 will lead to both sticky ends of the linearized vector binding to 2 different inserts and preventing linearization. 

After digestion are you heat killing the restriction enzymes? You should also do a phenol/chloroform extraction to remove excess enzyme and dilute residual digestion buffers that can affect ligation reactions. 

Additionally are you using old ligase buffers? The added ATP often hydrolyzes at room temperature after multiple thaws. I often add additional ATP to my ligation reactions unless using a brand new buffer. 

Are you ligating or PCR sewing your two constructs together prior to ligation? You should do this to ensure directionality (capable to do so with PCR) as well as increasing entropy variables in favor of your reaction. 

Dear Nhan,

First of all, thank you for providing with details of your experiment. I have made several constructs employing the strategy described by you. However, I do it little differently and it always works. Please try it like this:

PCR amplify the two fragments and purify them. Restrict the two fragments with XbaI and purify. Ligate the two fragments (50 ng each) in 1:1 ratio at room temperature for 60 min or at 16C for overnight.

Setup a PCR reaction; use forward primer (with EcoRI RE site) that hybridizes with fragment 1 and reverse primer (with BamHI RE site) that hybridizes with fragment 2. Use 1-2 uL of ligation mix as template. You should be able to PCR amplify the ligated product. Purify the amplicon, restrict with EcoRI and BamHI and purify again. Use this fragment to clone into the vector at the same sites.

Alternatively, ligate 100-500 ng of XbaI-restricted fragments 1and 2, purify the ligated product from the gel, restrict with EcoRI/BamHI, and clone in the vector at the same site.

Good luck.

@Jose: That's one option I've been thinking of. This may save my time a lot.

@Nicholas: I tried different ratios, varying from 1:1 to 10:1. The latest trial, as I mentioned, is 60:1 ratio because I found this suggestion in a similar topic at https://www.researchgate.net/post/Cloning_trouble_No_insert_in_colonies_that_form_on_ligation_plate_but_get_no_colonies_on_vector_only_plate-can_anyone_help

So far, I'm not lucky yet.

I don't do heat inactivation after digestion since it can affect my DNA. I either did PCR clean up, gel extraction or ethanol precipitation instead.

I used to use old ligase buffer. This buffer has been aliquot to small amount by previous lab technician. Moreover, for the safe side, I got a new ligase kit recently so I'm pretty confident the buffer in that kit is still in good condition.

I also tried ligating 2 fragments prior to cloning into vector. I saw the ligated band on the gel (at least in term of expected size). However, after gel purification, I don't have much left for PCR. Increasing ligation amount can be a solution for this issue.

@Syed:  I've tried your options but so far I'm not lucky yet. 

+ I myself don't think there's any issue with my restriction enzymes because I've got new RE recently and also checked their efficiency before use.

+ I think problem may come from the ratio used for this 2 fragments. I will try it again.

@Sandeep: I've never tried 10oC incubation before. Can you explain why it should be at this temperature?

Hi Nhan Huynh,

Syed's suggestions are excellent.

As further alternatives: check to see if there's an XbaI site between the EcoRI & BamHI. If so then clone one fragment first followed by the second.

If there's not and you want to order a new PCR primer for the 3' end of fragment one with has both XbaI and BamHI sites. This can the be cloned EcoRI - BamHI into the vector, then the XbaI - BamHI fragment can be cloned into  it (assuming on other XbaI sites).

-Richard

p.s. thanks for stating what you have done so well,  it's a real help.

Dear Nhan,

I forgot to ask but your assembled fragment happens to be of viral origin? Colony PCR amplifies the assembled fragment using insert-specific primers or vector/insert -specific primers? How strong are the bands compared to control (vector alone)?

Once we encountered similar problem; the colony PCR was always positive but cloned insert was lost upon plasmid amplification. However, we were working with HIV molecular clones.

I concur with Richard's solution. Unfortunately, in pUC19, XbaI lies downstream BamHI. You can clone your first fragment between EcoRI and XbaI but to clone 2nd fragment, you got to order a new reverse primer that hybridizes to 2nd fragment and contains any one of the RE sites that are present downstream XbaI in pUC19 MCS (and obviously not present in your 1st and 2nd fragment).

@Richard: thanks for your advice. I thought about it as well. Unfortunately, my vector doesn't have XbaI site between EcoRI and BamHI so I still still have to either ligate all fragments at once or ligate my fragments before cloning to vector.

@Syed: My fragments are fruit fly origin. For colony PCR, I used vector/insert specific primers. I included the control (Ctrl) in the last lane on the right side of attached figure. I don't see any band since one of the primer is sequence specific. In my point of view, the bands are real. My concern is maybe for some reason, the plasmid just flow outside of the bacteria instead getting into the cells. One possible explanation can be my competent cells are not as good as I believe and cannot take the heavy plasmid (in this case is about 8.4kb). However, in my other project, I work with another plasmid of around 9kb, using the same competent cells I made and everything was fine. E.coli still get the plasmid without any problem. So I'm still stuck at this point.

Dear Nhan,

Your bands appear legit. These bands represent the assembled fragment? What was your negative control? Whenever I do colony PCR, I use empty vector -transformed bacteria as negative control. This is to make sure that nothing is being amplified from either host genome or plasmid backbone.

Instead of growing the positive clones in broth, simply spread them on selection agar plate. Next day, scrap off bacteria and isolate plasmid. See if it helps. Also, please grow your transformants at 30C and not 37C. Finally, you may want to switch to some other bacterial strains such as JM109 etc.

Hi Syed, 

Those bands cover parts of my fragment and plasmid. I use a primer pair to check both ligation efficiency and direction of my insert. My negative control was PCR from colony transformed with uncut plasmid. 

Thanks for your suggestion. I will try again under those conditions you suggest. I will let you know if it works.

Hi, You are going to insert 2 fragments in a Particular way, i suggest not to use 3 or 4 RE, its better to use SOE PCR, its easier and save much time rather than keep incubating and purifying, once you constructed your fragment, you can t-clone it and use when ever you need, just cut from t vector and insert into you required one. I hope it will work for you,