Dear Roy,

Thank you for your answer. The plasmid also, has EcoRI site, means after digestion doesn't matter if it has insert or not, it needs to be linerized. That is why I don't think the ligation failed, but probably something is wrong with enzyme. With same enzyme for same samples, I set the reaction but this time my incubation time was O/N. I got desired band for insert (1,5kb) but the band was weak and the plasmid was again located above it's actual size. That is why I am also thinking enzyme lost it's efficiency and also concentration of plasmid needs to be decreased.

I think you should check the quality and the size of your vector and insert. When the insert and the vector run in the gel correctly, you should purify them, treat the vector with CIAP and then you can proceed with the ligation. If you still get the bigger bands after the digestion, you might have some contamination issues (colonies contain bigger vector with bigger insert?) because unsuccessful ligation should not give any colonies.

Of course, the most important thing is that the vector and the insert should contain complementary ends in order to get the correct ligation. There is no information about the ends of your DNA (which restriction enzymes were used or are there polyA and polyT tails involved? are the DNA ends sticky, blunt? etc.). But I am not familiar with instaclone PCR cloning kit and it might clarify some things.

Dear Teder,

Thank you for your answer.

I first designed my primer in a way that they contain EcoRI site at both ends than I amplified my insert and I got band at 1.5 kb (as I expected), later I isolated my insert from gel and than ligated my insert with 2.9 kb plasmid. InsTAclone PCR cloning kit allows you to do TA cloning so I basically ligated my insert (generated with Taq pol. (3'A)) with my plasmid which is 5'T (so I did ligation without restriction enzyme reaction).

That is why I do not think that I have contamination in my insert, because I already isolated it from gel.

As you can see the picture below I again did restriction enzyme digestion. First band (i do not count ladder lane) is my plasmid without insert and BamHI cuts it once and I got band at 3 kb (as expected). In lane 2 and 4 I digested plasmid with EcoRI and was expecting two bands but obviously something went wrong. But the strange thing is in lane 3 I cut construct with BamHI (which is single cutter) and got band between 8 kb and 6 kb. I am wondering how is that possible. I know it is a bit crazy to think that but when you look at lane 3 (Plasmid with insert BamHI) we can say that it is located at almost 7.5 kb and if my insert somehow enter between two plasmids and those two plasmids somehow circularize, the new size almost will be 7.5.

I am wondering your answers,

Best.

Dear Fukan,

from a quick look at your last gel, I would conclude that:

1. EcoRI does not cut your plasmid. Compare lines 3 and 4. BamHI can obviously linearize your plasmid. It is not clear if EcoRI is cutting at all, what you see may be uncut, circularized plasmid (tip: next time load some uncut plasmid next to the EcoRI digested. It would help understand if your enzyme works or not).

Conclusion 1. Either the map of your plasmid is wrong (point mutation in EcoRI) or your enzyme does not work. Suggestion: test the enzyme on a different plasmid. Load uncut plasmid next to it to check for actual cleavage

2. Your positive clone is double of the size of the original vector (lane 1, lane 3), it has a single BamHI site, and it is not clear if it has EcoRI sites at all (I bet not, see point 1). This is coherent with the vector ligating to itself, without taking any insert. That's pretty common.

Conclusion 2. Your positive colonies most probably DID NOT take the insert, but you have instead ligated together 2 plasmid backbones, as you anticipated. Prediction should be that a restriction with EcoRI would produce 2 bands since now the restriction site is duplicated. I can't see that in your gel, but it is clear that there is something fishy with EcoRI (see point 1). Suggestion: digest again the plasmid with a RE that is unique in the original plasmid (and that is not EcoRI or BamHI), you should finally see a double band. The size of those two fragment will tell you if the insert was taken.

Final conclusion: you would probably be sure that your EcoRI is still active and that your plasmid has the correspondind sites before using it to draw conclusion (can you linearize the original plasmid using EcoRI?). In any case, the insert was not uptaken. This is likely to be to poor insert preparation. Suggestion: prepare your insert again and check again your primer design. Did you add some extra nt on the BamHI primer to ensure that there is enough space for BamHI to sit and cut? (https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments)

Dear Tanino,

Thank you verymuch for your explonation,

I isolated 12 more colonies from the same ligation and cut all with BamHI to linearize the vectors and as it is seen in figure 1, I loaded them on gel to see their sizes. As you can see there are 6 constructs that has the size around 7.5 kb (I am not complately sure how is that possible). Than 2 plasmid located at the place of 3 kb (those are plasmids that did not take up insert), than there are 3 constructs that did take up insert and located around 4.5 kb (insert=1.5kb plasmid=2.9 kb).

Than I took those constracts which located at 4,5 kb and digest them with different EcoRI and got the result in figure 2.

I got the expected results, the only thing that I am wondering is that as you can see in the figure 2 one insert (first one in figure 2) gave band around 100 bp smaller than 1.5 kb.

Regards,

Good to hear that.

Send them for sequencing and check that everything is alright.

Concerning the shorter insert (1,4kb), it may come from aspecific amplification of your primer (then, the insert is not what you expected to be. Do you see any 1,4kb band on your original PCR?) or partial degradation of one end of your PCR product (not sure if this is possible in your specific case since - if I have understood your cloning strategy - then this should cause either BamHI or 1 EcoRI site to disappear).

In my experience, it is not surprising that after the digestion of your ligated constructs (especially while using instaclone kit), some of the samples might give unexpected fragments. As you already realized, you needed more than 5 colonies to sort our the correct constructs. I usually start with 12 colonies and also might repeat the ligation or transformation with the ligation mixture or even repurify my PCR product if I am still getting negative results.

I agree with Tanino that you should sequence potentially correct constructs. It gives you an idea what's really in your tube.

I randomly sellected 12 colonies and got 3 expected constructs. Before sending to sequence I randomly selected 12 more colonies to increase the sample size for sequencing and 12 colonies gave band at 7.5 kb (expected size is 4.5 kb). It was really weird, somehow I cloned more than one insert in TA cloning but I dont know how is that possible.

Some quick math:

Vector (digested EcoRI-BamHI) = 2,9kb

Insert = 1,5kb

Desired ligation event: 2,9kb + 1,5kb = 4,4kb

Double insert: 2,9 + 2x 1,5kb = 5,9kb

Triple insert: 2,9 + 3x 1,5kb = 7,4kb

Detected plasmid size (non desired products) = approx 7,5kb, matching the prediction for triple insertion.

Maybe you have such an high molar ratio of insert over the plasmid, that inserts ligate between them before being ligated to the plasmid.

This is " Maybe you have such an high molar ratio of insert over the plasmid, that inserts ligate between them before being ligated to the plasmid " exactly what I was thinking. I am just wondering the mechanism of it because theoretically it is not possible.