We are using pJET1.2 vector (Thermo CLoneJET PCR Cloning kit) for cloning 1400bp fragment. We have blunted our insert following kit protocol. According to kit self ligated vector should not grow on plate due to lethal gene. But we are getting 8 out of 10 colonies to be false ligated. Can anyone please tell what can be the possible reason? We have used insert:vector in 3:1 ratio. Gel image after colony PCR is attached.
We use this kit on daily basis...It seems you may have one of the following problems;
1. Best is to blunt the PCR products and then elute after Agarose Electrophoresis as non purified PCR product may have primer dimer or small amount of non-specific products that ligate to give false/non-desired colonies.
2. For better results, amplify by proof-reading enzymes, run the gel, elute and ligate.
3. Your pJET1.2 vector may be old/degraded a bit so that frame of the enzyme that kills normal E. coli changes.
Dear Sukanya,
Why don't you try an alkaline phosphatase treatment. That should help in your case.
Abhishek
Maybe the problem is in your colony PCR. Do minipreps of colonies transformed with ligation reaction and digest the inserts out of the vectors. This will tell you better if you have self ligation or not. Also make a ligation reaction with digested pJET vector only (no insert) and see how many colonies will grow.
This is one of the best kit for initial cloning. There is no problem of self-ligation because this vector contain self killing gene. Apart from that this is highly efficient kit and give approx 9/10 colony positive in cPCR. In my opinion use following step.
1. Use proof reading enzyme as it also give the blunt end so it reduce your one step.
2. Standerize your PCR, I didnot consider this band as positive because cPCR band is always good . I guess your band is approx 1 Kb if longer than 5 kb plz try primer from middle section. Always Run the negative (no template) and positive (pure plasmid) control with PCR.
3. No need to use any alkaline phosphotase and give atleast 20 minute for ligation.
4. It may possible you comp cell got contaminated so streak them on antibiotic agar plate and check the status.
5. If problem persist use the single F and R primer in different tubes for amplification of product (for ligation) sometime single primer gave the band with crude DNA but not in specific condition e.g. cPCR.
Hope for the best. Thanks
Thank you for the replies. The band shows same length if amplified using pJET primers in an empty vector. Everything is working fine if we use Promega pJEMT vector so I guess competent cell is not the problem.
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