To carry out blue white screening for confirmation of ligation of PCR products into desired cloning vector
Ecoli strains (DH5α), PCR product, T4 DNA ligase enzyme, Luria Bertanni agar, Petri plates, ice, double distilled water, X-Gal, pipettes, PCR master mix, desired restriction endonucleases (Nybo, 2010).
Bacterial colonies were obtained which were mixtures of blue and white colonies. Presence of a positive clone will be indicated as a white bacterial colony since the beta Gal in the vector will be disrupted due to the insertion of the gene of interest. Hence there will be no functional beta Gal to metabolize X-Gal (Yuanxin, 2003). On the other hand, negative bacterial colonies will have intact beta Gal that will metabolize X-Gal producing blue bacterial colonies.
As per observation majority of the bacterial colonies were blue except one colony that was white. Also two colonies were observed that were found to be partially white colonies. This suggests us that the positive clones containing the gene of interest might be in one of these three colonies. Hence for the purpose of further confirmation the following needs to be done:
The above set of experiments indicates that the cloning of the gene of interest into the desired vector was successful. However further experiments (as mentioned above) needs to be carried out to absolutely confirm the cloning success.
Nybo, K. (2010). DNA and General PCR Methods: Blunt-end Ligation. Biotechniques, 48(4), 273-275. https://dx.doi.org/10.2144/000113397
Theophilus, B., & Rapley, R. (2011). PCR mutation detection protocols. New York, N.Y.: Humana Press.
Yuanxin, Y. (2003). T-linker-specific ligation PCR (T-linker PCR): an advanced PCR technique for chromosome walking or for isolation of tagged DNA ends. Nucleic Acids Research, 31(12), 68e-68.
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