Dear Ruojin Tian,

I have seen these positive results before from colony PCRs when you use the colony directly from the transformation plate. If you are using primers inside your insert and a good DNA polymerase, you can amplify your insert from the fragments of DNA that are spread over the plate, not inside the cells. This DNA comes from the ligation you used to transform the cells and the colonies that you obtain contain a empty vector.

To solve the problem with the colony PCR, you should use primers located in the vector that produce amplification of your fragment. If the fragment is really long, you can use a primer inside the insert and other in the vector.

Concerning the toxic expression of your insert, do you know if the expression is due to your vector or is your insert which is been recognized by the transcription machinery?

Colony PCR is not reliable. Use freshly made LB+Antibiotic plates, streak the colonies obtained, and do mini prep with well grown streaks. Your PCR and Rest Dig results are final

Dear José Tomás Cánovas-Márquez

My gene is a recombinant one, there is one part that comes from the plant, one part comes from the bacteria, another part is coming from the virus. I used the toxic gene prediction website https://exploration.weizmann.ac.il/perl/pandatox1_0.mperl?submit=blast

but I am still trying to understand the result it provided since none of the genes from my insert has been recognized as "non-clonable".

Regarding what you mentioned, my promoter is a rubisco one, it might somehow triggered the transcription and the protein damaged the e. coli.

Thank you Papri Basak

My question is there not supposed to have colonies that contained empty plasmid, cuz the restriction enzymes recognition site does not share the same sticky end.

The only chance could be the plasmid I transformed failed in the digestion and they are the original ones, but the gel I ran indeed shows good linear bands around the right size.

I am so confused.

Hi again Ruojin Tian

Considering the information you wrote, It seems for me that first you have to discard a problem in the ligation. You are using high efficiency competent cells which means that if your ligation is not working, even a really low and undetectable amount of undigested vector will yield resistant colonies after transformation.

When I have this type of problems:

1) I review all the DNA maps, including PCR primers, and simulate the construction to be sure that all the restriction sequences are present. When I introduce the restriction sequences by PCR I add at least 5 additional nucleotides before because some enzymes don’t cut properly in the end of the DNA fragment.

2) If everything is properly designed, I check the compatibility of the enzymes for double digestion. If one digestion is “difficult” because for example I need to use 4 units of one enzyme for each unit of the other (low activity in the most compatible buffer for double digestion), I perform a single digestion of the vector with the low activity enzyme in this buffer to be sure that is working.

3) After an overnight digestion of the vector and insert, I purify both from gel. This step is really necessary when the buffer contains high amount of salts that will disturb the ligation.

4) In cases as yours, I also perform a fosfatase treatment of the vector despite in theory is not necessary. I saw that when the ligation is hard to obtain, with this treatment I can eliminate the low percentage of vector digested with only one enzyme.

5) Finally, I perform the ligation with no more than 50-100 ng of vector and the corresponding molar amount of insert.

If the problem is in the ligation, doing this checks you will succeed, but if the problem is due to the leaky expression or others you won’t obtain colonies in your plates and you should change your approach as trying other vectors or working with linear fragments, doing your construction directly by fusion PCR.

José Tomás Cánovas-Márquez thank you very much for such a detailed and encouraging reply.

I want to ask you another thing, I actually also tried to digest and ligate three fragments directly to pCAMBIA and express in Agrobacterium. I checked the pCAMBIA, as a binary vector, which should be good to deal with the situation of leaky expression.

Regarding digestion, the enzymes I use is from Fastdigest which instruct me to do the digestion within 5-15 min. Will the O.N. digestion enzyme be better?

I will also do a negative plate next time (only the cut vector purified from gel) to see if there is actually original plasmids.

I also learn few ways to do the ligation, one system is digestion+ligation in the same time, another is one step cloning that will be no need for digestion: ClonExpress II One Step Cloning kit.

Dear Ruojin

I am not sure to understand your first question. If you want to perform a three-fragment ligation you only need to be scrupulous with the design, the selection of the restriction enzymes and the equimolar amounts of the inserts. But the binary vector system you talk about is used for plant infection and integration of DNA mediated by Agrobacterium. If you are planning to transform Agrobacterium directly with the ligation to obtain your plasmid, this could overcome the toxic issue but won’t work if the problem is in another step. I found this paper that you can check to think about this option:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5821610/

Regarding digestions, if you use Fastdigest enzymes you won’t need to perform overnight reactions. In fact, you can have star activity if you extend the reaction time. Only be sure to don’t overload the reaction with an excess of DNA and inactivate or purify the fragments after digestion. Think that if you have an excess of substrate your reaction will render a mixture of fragments digested with one enzyme, the other or both.

It’s a good idea to introduce the negative test. I always do it for each new preparation of vector that I produce, but I understand that usually we only do it when we have problems. My Ideal situation to produce a really complex ligations of multiple fragments is when the phosphatase-treated vector produces 0 or 1 colony in the transformation. This ensures me that even with a low chance to obtain a construction, if everything works I will obtain it.

There are many approaches to build your constructions depending on the money or time you want to spend. I haven`t tried one step digestion+ligation, but when we need large constructions that requires several steps of restriction and ligation we usually use in fusion cloning kit (Takara) that is similar to Clonexpress and works really well.

Try to divide the cloning in multiple steps if possible using unique restriction sites in your insert if present and adding in bits (2 or more). Alternatively, try adding the promotor later than the insert if possible.

José Tomás Cánovas-Márquez thank you. I bought the infusion cloning kit, also the kit for cloning PCR in agrobacterium. I hope it will work, and I'll keep trying repeat the experiments with the vector ligation only. I also found my primers could be problematic, so I already ordered new ones. To discuss with you is really helping me a lot. It straighten my thoughts and encourage me to keep trying and found the best solution. Thank you!

Kafeel Ahmad thank you for your reply, I will try the method you suggested

Hello guys, after I retrospect my experiments, I found there are few things I did it wrongly: 1. there is a chance my primers not really working; 2. my ligation is not really working; 3. the plasmid was missing during the regrow of the starters of the colonies; 4. I did not do the transformation with the digested vector only (could be my vector is partially digested, could be there are many false positive)

so I decided to repeat my experiments including the solutions for all the mis-happens.

Hope the answers can help you.

It seems you're trying to clone a gene that E. coli does not like. So it will rearrange.

Either screen more candidates or else try to transform directly your sequence (ORF) with homology arms into tobacco.

Holmes Allyson hi, it's very intriguing what you suggested! Could you please tell me more?

Your question and problem is not trivial.

Perhaps this paper will help:

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene gun....

Mattozzi et al., 2014, Jove

https://europepmc.org/backend/ptpmcrender.fcgi?accid=PMC4172236&blobtype=pdf

So there's no Agrobacterium middle step here.

and also this vector pAU10...

Article Development of a novel vector for cloning and expressing ext...

And you can find the Gibson Assembly Cloning approach here as well:

https://www.addgene.org/protocols/gibson-assembly/

I also suppose you could use CrispR/Cas9 as well to transform proptoplasts:

Cas9-based genome editing in Arabidopsis and tobacco

doi: 10.1016/B978-0-12-801185-0.00022-2.. 2014;546:459-72.Methods Enzymol

Holmes Allyson thank you for the suggestions. I will check the papers carefully!