Another reason, different to standard molecular cloning issues, is the possible toxicity of the gene inserted to the host. There are evidences and tools to predict/overcome that condition. A lethal product for the host is the possible explanation of genomic regions with very low coverage representation in previous Sanger shotgun genome projects dependent of plasmid gene libraries.

http://www.genomics.arizona.edu/553/Readings/Sorek2007.pdf

http://genomebiology.com/2013/14/5/R51

http://www.ncbi.nlm.nih.gov/pubmed/22705841

Hi Victoria,

one reason I can think of why your cloning doesn't work might be the sequence you try to clone. If your fragment contains repetitive sequences it might be that the used E. coli strain is recombining it out of the vector. Many common E. coli lab strains (like DH5a) are already reduced in their recombination ability. However this sometimes still enough to cause trouble. For such cloning approaches we usually use the Stbl3-strain which is further reduced in recombination ability.

Best wishes,

Andreas

Thank You Andreas. I will try cloning in a different strain

Hello Victoria!

In our lab we use the Promega pGEM T-Easy Vector Sistem I as a cloning vector. I've already cloned up to 2.5 kb fragment in this vector.

A reasons I can think about your problem is that your PCR product don't have a adenine tail. You can solve this by adding a 5 - 10 minutes Final Extension step.

It's important to fallow all the recommendention in the protocol (e.g: vector : insert ratio).

We use DH10B as strains for cloning. It works perfectly.

Good luck!

Luiz

Another reason, different to standard molecular cloning issues, is the possible toxicity of the gene inserted to the host. There are evidences and tools to predict/overcome that condition. A lethal product for the host is the possible explanation of genomic regions with very low coverage representation in previous Sanger shotgun genome projects dependent of plasmid gene libraries.

http://www.genomics.arizona.edu/553/Readings/Sorek2007.pdf

http://genomebiology.com/2013/14/5/R51

http://www.ncbi.nlm.nih.gov/pubmed/22705841

May be ligation is not working. May be you need to optimize that. Secondly try PGEM-T Easy vector is a good vector may be use that one. I have used it to clone fragment of almost 1.8 kb and process is quite easy too. Also use JM-109 cells for bacterial transformation. Regards

Victoria,

Check to ensure that your products are properly ligated. After you have completed your ligation reaction, take a small amount of the reaction and perform PCR using primers that flank the cloning site. You should generate a PCR fragment that is 2kb + the length of the primers and distance from the cloning site. You may also generate short fragments produced from re-ligated vector – this can act as control for your PCR. If there is no PCR product of the right size then consider how you isolated and prepared your fragment. I suggest that after PCR or a restriction enzyme digest, load half (or less) of your products onto a gel to view the results. If you get a single sharp band on the gel, clean the PCR products that you held back (lots of kits are available) and then use that DNA for cloning. The DNA purity will be much higher than you will get from purifying DNA from agarose and will thus greatly improve the efficiency of subsequent cloning. If you have to cut a band out from a gel, clean it up carefully before cloning. Consider alternative cutting sites or enzymes. If possible cut your vector with different enzymes that generate non-compatible ends so that the possibility of vector religation is minimized, your fragment of course will have to be compatible with those ends. Fragment instability and product toxicity are occasional problems, as has been pointed out by other contributors, but check the initial steps of your cloning reaction before investigating too deeply into those other possibilities.

Some good answers there, but remember that extended palindromes are not stable in most E.coli strains.

I read a poster many years ago about cloning of chloroplast DNA sequences and one just wouldn't work. Turned out it had considerable sequence similarity to OriC so the plasmid became subject to stringent copy-number control. Sorry I've forgotten who the authors were: it was a nice piece of work.

neither of these are lethal ti the host cell, but properties of the sequence you are trying to clone.

It is just not a possibility, but it is a fact. Try mismatch repair technique and then clone.

If the DNA sequence is repetitive (especially inverted repeats) then you may not be able to clone it into a standard vector - use a lower copy plasmid such as pBR322, and try growing your colonies at 30 degrees instead of 37. Also, try an overlapping recombination method (eg InFusion or Gibson Assembly) as that may be cleaner than T cloning.

One of the possibilities is that the genes to be cloned expresses a toxic product, for instance, some membrane proteins, the cloning can be very difficult if the vector giving high expression. If this is the case, it is preferable to use a vector which the promoter is tightly regulated or one's expression can be suppressed.

Since you are trying to clone a membrane protein, which is very similar to what I had experienced, I think the chance of toxicity problem is quite high. You can try to incubate the plate at perhaps 15C instead of 37, that sometimes can help reduce the toxicity, or pick a different vector.

One of the way we clone such large fragment unless it is toxic to the E.coli, is to first codon optimize for E. coli and have appropriate restriction sites that are not present within the gene, then get it synthesized. It used to be very expensive but now a days there are companies that guarantee such constructs in E. coli. It is well worth the cost if you absolutely have the construct.

If it is toxic and by the description it looks like the insert is being rearranged to make it non-toxic. Some of the low temperature incubation and minimal media may help.

all the best

There are a number of possible reasons. As well as those mentioned above, repeative DNA sequences within the insert can cause DNA to fold on itself and become difficult or impossible to clone. I suggest you proceed by cloning the sequence in bit parts (sub-fragments). Hopefully chopping up the full length fragment will disrupt what ever it is that is causing problems. Good luck!

I know Dr. Venter and his colleagues have replaced an entire genome of mycoplasma with a synthetic new genome of another mycoplasma species. But any one has been able to clone the origin of replication?

Hello, I agree in the two possibilities that either ligation is not successful or that the product is toxic to the host.