Size is not the problem, 5.5 kb is not that large unless there's something weird going on regarding the sequence of the insert itself. Those vectors often come in cloning kits with positive controls, etc.; have you tried running a control reaction?
If it's only for cloning i would choose pBlueSK.. And maybe let the bacteria grow at 32°C instead of 37°C. We had also problems with huge constructs and unwanted recombination. Once i also tried different bacteria. There are bacteria for large constructs such as targeting vectors avaiable! Good luck with that!
Can you give us some more details on what you tried? I cloned a couple of genes in the same size range with pJET and it always worked.
Either of the plasmids you tried should be fine for a 5kb insert. I suspect you are having other technical problems that are not size related.
Hi Maximilian
Actually I tried a 3:1 insert vector ratio and transformed into dh5alpha cells using transformaid kit. I got colonies, but they did not had plasmids. I am a bit confused how this happened.
Did you column or gel purify your PCR product?
It is impossible to guess what went wrong if you don't write what you did (with all the details).
Hi Maximilian, can you give your protocol of cloning using pJET vector
It would be a great help for sure
The gene of interest was amplified from cDNA in an initial 20 microL PCR reaction, most of it was run on a agarose gel, if they band was clearly visible 1 microL of the initial reaction was used for another round of PCR (100-200 microL), the reaction was column purified, ran on a 1% gel, eluted with a minimal amount of elution buffer and then used a described in the manual of the pJET cloning kit. We use Phusion for the PCR and columns from Macherey-Nagel.
The DNA has to be gel purified (or something similar with stringent size cut-off), any primer-dimer, small size amplicon, whatever, will kill your cloning step. The ligation is much more efficient for small double-stranded DNA, so anything smaller than your gene will enter the plasmid.
Size should not be a problem with the vectors you are using. Use 100 ng insert and 550 ngm vector (pTZ57R/T that you are using). I hope you are doing tailing of your purified insert with dATP if using TA based cloning kit. Keep it overnight at 16C and preferably use electroporation instead of heat shock. Use fresh competent DH5alpha cells. Parallel to it, take a positive control for checking the (i) efficiency of cells and (ii) effiicacy of ligase. I am sure it will work unless your protein encoded by your cDNA has some lethal effect. All the best.....
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