What about using adapters containing the cohesive end you need?

For the PCR ligation... there could be many reasons, but none of them related to the presence of restriction sites. If using Taq polymerase, the enzyme adds an -A at the 3' end of the molecule, thus for efficient cloning you need a vector with a -T overhang. If using Pfu / Phusion, that shouldn't be a problem, since the PCR product has blunt ends. Just remember that primers are not phosphorylated, and so will be your PCR product. So, if trying to clone that into a dephosphorylated vector, it simply won't work.

Definitely I prefer the Gateway system for cloning, since you are not limited by the availability of restriction sites, so it is easier to control what part of your sequence is included into the vector. It also allows you to use the same PCR product for cloning into any vector based on this system. If you already have primers designed for amplifying your sequence, you only have to order new primers with the same sequence adding the recombination sites needed (which are standard for all Gateway vectors). Alas, you will also need to purchase the Gateway vectors, but it is worthy.

Gibson cloning is probably a better choice than Gateway for your purposes since the latter adds att recombination sequence which may not be suitable for your purposes. Several commercial products are available (NEB Gibson, Clontech In-Fusion, Invitrogen GeneArt) which all work well, but typically cost $20 per reaction. These techniques rely on homologous sequences between your PCR product (incorporated into your PCR oligos) and the linearized vector of interest.

However, I wouldn't give up all hope on the restriction enzyme approach. If you provide some more info on your vector and the source of your cDNA insert, I can offer more specific advice on how to proceed. Can you post the maps, or sequences?

I agree with with Gustavo. I just will add to the above that if you are using a special plasmid you will need to make it an entry vector for the gateway technology. That means you need to buy the gateway cassette from Invitrogen together with the pDONOR and the clonase. You will have to perform blunt-end cloning. I have done that longtime ago for a fission yeast specific plasmid. It is worth if you and your colleagues in the lab intend to use it regularly because it requires a bit of investment.. If you are using E.coli or even mammalian cells you have plenty of gateway vectors to choose from.

Alternatively you can add more restriction sites to your plasmid by adding a double stranded oligo. Otherwise you can perform blunt-end cloning.

I agree with Daniel. Gibson cloning is very user friendly.

I am still following the protocol in:

Enzymatic assembly of DNA molecules up to several hundred kilobases.

Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO.

Nat Methods. 2009 May;6(5):343-5. doi: 10.1038/nmeth.1318. Epub 2009 Apr 12.

I make my own mix from individual components. I think it is cheaper than any of the currently available commercial kits.

I uploaded my answer before Daniel’s posting. Yes, Gilson assembly is convenient, though up to now I’ve never tried and people are having problems when using multiple fragments. Not sure if Gilson's approach allows for circular assembly. If yes, then one may need to sequence quite a lot to discard the introduction of mutations not only in his insert but also in the plasmid. You may argue that it is functional, etc...

but I am not sure if it is worth to invest such efforts in a routine cloning experiment.

Adbelhalim- Gibson assembly does not require PCR amplification of the vector. The requirement is 15-20 overlapping homologous sequence at the 5' and 3' ends. This requirement can be completely satisfied by designing appropriate PCR oligos for the insert, and linearization of your target vector using appropriate restriction sites. On technical grounds, it is just as reliable and efficient as REN-mediated cloning. Personally, I only recommend REN mediated cloning if direct subcloning is an option (obviating the need for PCR amplification of the insert) and/or if cost considerations preclude the use of Gibson or other recombination-mediated technologies.

Thanks Daniel for your clarification!

Daniel and Abdelhalim- both Gibson and Gateway introduces nucleotides at the ends of the primers and this at least for Gateway gets into the plasmid. Obviously, since the ORF is maintained one will expect for the case of say over-expression, that should not be a problem provided these nucleotides do not affect rRNA or other important and conserved regions of the plasmid required for its functional properties. Is that right and do you supposed this might occur. l am presently trying to shift totally to a more efficient system and although very costly Gateway at least, l will like to know the bottlenecks before l make the huge investment. Thanks

All these methods have their trade-offs, so there is no singular ideal solution for all cloning needs. If I were to recommend one method for you, I'd need to know more about what system you are using (bacterial/yeast/mammalian or other) and what your goal is. You reference rRNA (ribosomal), but perhaps you mean mRNA? If you are willing to map out more details on your experimental design, and the sequence info for your target DNAs (insert/vectors), then I could make a more specific recommendation for how best to proceed.

To my knowledge and as far as I recall those sequences are not translated. So don’t worry about them. The advantage of using gateway is the possibility of quick shuttling constructs between different gateway compatible vectors without the need of sub-cloning. It is possible to test different tags, switch between species, etc….Beside this, I don’t see any. I wouldn't do it just to avoid regular cloning….I don’t see where is the complication or the issue in your case. Anyway, Daniel has kindly offered to provide you more detailed way of doing it if you provide more information.

I agree with Daniel. Every method has a unique tradeoff and that is why your cloning need determines what method should be followed. Your direct PCR ligation would never work because of the fact that you are trying to ligate sticky ends with blunt ends (unless the restriction enzyme you selected gives you blunt ends)

Since you are ligating a 2.7 kb product ino a 6.2 kb vector, you could try the following:

Use round the horn amplification technique to amplify your plasmid (http://openwetware.org/wiki/%27Round-the-horn_site-directed_mutagenesis). Leave the mutation part out from the protocol since you dont need to make any mutations but do use phosphorylated primers, DpNI digest the amplified linear vector and ligate it with your gene of interest. Transform and screen for the right orientation of your gene. Good luck.

Thanks all for the answers. Daniel-Please see attached the plasmid. l want to replace the expression cassette with my gene. Any suggestions will be welcomed. Thanks again

I think you don’t have the original plasmid. Am I right? In this case you well need one. If you have the restriction map of the original plasmid I would take out the GFPx cassette with HindIII/ BamHI double digest and restore the original MCS was it only for future cloning. That will take you only few days. There are other possibilities but I think this is the best and it is practical. Maybe Daniel is working out something else

Ok. So HindIII is a problem at the 5' end because it is present in your GOI. What about the 3' acceptor site? It appears the vector has XbaI and BamHI 3' to the GFP cDNA. Are these excluded from your cDNA? If not, then the best solution is to try Gibson cloning.

If however, these 3' acceptor sites are OK, then you simply need to find a suitable 5' site. If that's the only hurdle, simply order short phosphorylated oligo adapters with a 5' HindIII site and a 3' end that will match an overhang of your choosing for your cDNA. Alternatively, you can gap-fill the HindIII site on your vector using T4 DNA Polymerase and do a blunt end ligation at the 5' end. Make sense?

I think better than performing blunt end cloning. He can make HindIII compatible with XbaI (NheI, etc…) by fill-in with 2 nulceotides and the polymerase. (Hind III end with dATP+dGTP) for the vector and (XbaI with dCTP+dTTP). for the insert. That leaves him with two bases overhang, which is better than blunt end. For BamH1 he has several isoschizomers to choose from. It is important that first you carry the fill-in first and then perform the second digestion with BamH1 on the vector and the isoschizomer of your choice on your insert.

Hi Daniel. Not to complicate things too much, but another easy option is to mask the HindIII site in your insert by doing a 3 piece ligation. The strategy depends on how many internal HindIII sites you have, and where they are located. However, assuming only one internal site, you can amplify your PCR insert in two parts, as follows:

5'HindIII-1st segment of your cDNA-3'site (unique internal REN site), 5'(same unique internal REN site)-2nd segment of your cDNA-3' XbaI or BamHI site. This will allow you to replace GFP and still perform directional cloning, assuming I understand your strategy and map info correctly. You can do a 3-piece ligation with your two digested PCR fragments and a HindIII-XbaI or HindIII-BamHI digested vector.

Thanks everyone. We have decided to go in for Gateway cloning for the complicated ones such as these whilst l still use the basic restriction digest cloning for those less difficult. However, l have learnt a thing or two and l still hope to try out the method of tailing the genes with nucleotide. Hopefully, it will work. l do use that for amplifying my genes in pGEMT but never thought to try that for direct ligation. Thanks again

Daniel- just to clarify, Gateway requires a special destination vector. If you need to stick with this particular vector (pRPA), then you'll have to do some cloning work first to make it Gateway-compatible. Alternatively, maybe you can find a Gateway destination vector that has already been made and suits your purposes. With Gibson cloning, you only need linearized plasmid. So this might be better.

Yes Daniel, we have just invested almost a £1000 in the system. However, the idea is to use the system to generate several gateway vectors for use by the group and other members in the university as well. It is definitely a very expensive venture. l hope to look more critically into the Gibson cloning system. Probably it will be cheaper and definitely it looks much simpler. Thanks again

The ability to easily shuttle cassette between different plasmids is a huge advantage of Gateway cloning, and fortunately there are some ways to keep costs down on the system. For one, you only need 1-2 microliters for a transformation, so you can scale down the volumes of the recombination reaction 2-3 fold. Second, you can multiplex the recombination reactions in a single tube, and screen for different inserts or different vectors using colony PCR. Lastly, for the really savvy, there are bacterial strains that will perform the recombination in vivo, but you need to perform serial conjugations, so while much, much cheaper, more labor intensive. Good luck!