Hi, your expectations are normal for the pJET cloning. When I cloned PCR products in pJET I plate something like 25-50 µl of the transformation and the plates are full with colonies on the next day. Unfortunately there are false positives because the lethal restriction endonuclease gets inactive once some bases are ligated to it. So you have to check some colonies to see if your product was ligated in the vector. I check 6-8 colonies and most often I have some positives to continue (around 50% depending on the ligated product). To be fast in checking colonies I use a nice Kit "Colony Fast-Screen Kit - Restriction Screen". There you pick your colonies on a new plate and in 10µl of the Restriction solution. You just cook the cells, add your restriction enzyme, incubate the samples and analyze them on agarose gel. And with the plate of your picked colonies and your results from restriction you can easily go on.
Hello Laura,
I have used the pJet cloning kit and according to the instruction prepared the transformation volumes....however depending on the nature of the insert the transformation efficiency may vary. I have had experiences similar to yours as well as cases where i get barely 10-20 colonies on LB-Amp plates. In case of abundant colonies I too take 6-8 colonies to verify insert. One way to avert this is vary the ratio of the insert to vector during ligation. Could you tell me what competent cells are you using for the transformation?
In cloning, vector/insert molar ratios is important. Please ensure that you are sticking to correct molar ratio. The kit suggests to use 1 uL (50 ng) of vector, which, according to my experience, can safely be used at half its concentration i.e., 25 ng. The kit suggests to use 1:3 (0.05:0.15 pmol ends) vector/insert ratio. Instead of one reaction using 50 ng vector, use two reaction containing 25 ng vector and try 1:3 and 1:1 or 3:1 vector/insert molar ratio.
This cloning system most likely makes use of ccdB gene that is lethal to cells upon expression. We have done some experiments with this gene and always found some population of cells to be resistant to CcdB-mediated toxicity. The sacB gene worked better in our hands as positive selection marker though it requires the presence of 5% sucrose in LB medium without NaCl and room temperature incubation.
Those who may be interested to find out more about this cloning system, following is a good read:
http://www.biotechniques.com/multimedia/archive/00011/96212pf01_11480a.pdf
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