Question

Please help. Thanks.

• From your libraries of clones, how do you use colony hybridization to find the “correct” clone?  Discuss the steps you would use.  You are attempting to clone novel kinase genes using the DNA from the protein kinase B [PKB] gene’s kinase domain as your probe.

Answer 1

Answer - So, Colony hybridization a technique in which we can clone our desired gene of interest and can isolate it from a colony of cells.

There are few steps for isolating the desired gene :-

1. Firstly, take master plate with colonies of bacteria( E.coli) with desired fragment of DNA.
2. Transfer these colonies on to the nitrocellulose membrane. because this membrane is rigid as compared to the master plate and can easily be used for this reaction.
3. Then use alkali treatment on this nitrocellulose membrane for lysis of the bacterial cells.(NaoH) which is a detergent. This is done because we want to isolate DNA from it.
4. Then DNA from these cells gets attached on this membrane and fixed at place by UV light. And heating is done at 80 degree celsius.
5. Solution containing radioactive probe is added.Which has a selective probe(Protein kinase B kinase domain) for the desired DNA fragment and complementary to it.
6. DNA hybridization with a radioactive probe attached to the sequences which are complementary to each other.
7. Subject this membrane to the X- ray (Autoradiography). For visualization of the hybridized probe to DNA fragment.
8. The final step include matching the hybridized DNA location to the colony of the master plate . And then we can isolate this colony and selectively grow this colony for kinase gene of interest.