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Question:

Discuss the purpose or aim of the experiment.

 

Answer:

To carry out blue white screening for confirmation of ligation of PCR products into desired cloning vector

Material:

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).

Procedure:

  • Gene of interested is amplified from the available source (Genome and or vector containing insert) using specific set of forward and reverse primers. The primers should have the desired restriction sites which must be same with that of the vector in which the PCR product needs to be ligated. Amplification was confirmed by running a 1% agarose gel.
  • Both the PCR product and the vector (in which the cloning needs to be done) were digested with the specific set of restriction endonucleases followed by elution of the respective digested products. Digestion products were confirmed by running a 1% agarose gel.
  • The eluted PCR product and vector were ligated using the T4 DNA ligase. The ligation reaction was carried out at 16áµ’C overnight. Ligation was confirmed by running a 1% agarose gel (Theophilus and Rapley, 2011).
  • The ligation product(s) were transformed into the specific E. coli strain followed by plating of the transformed cells into Luria Bertanni agar medium. The LB medium was supplemented with X-Gal for blue white screening of the bacterial colonies. Transformation of the bacterial cells with the ligation product was carried out under sterile conditions inside a horizontal laminar air flow cabinet.
  • The plates were incubated at 37áµ’C overnight.
  • After overnight incubation blue white bacterial colonies were observed in the Petri plate.
  • Colony screening was carried out by picking the desired white colonies with a sterile toothpick and was incubated in LB media overnight at 37áµ’C.

Observation:

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.

Results:

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:

  1. Plasmid isolation from the individual three bacterial colonies.
  2. Restriction digestion of the isolated plasmids using the restriction endonucleases to check for the fall out of the inserts (agarose gel electrophoresis should be used for this).
  3. For final confirmation sequencing of the plasmid samples should be done (Nybo, 2010).

Conclusion:

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.

 

References

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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