We plate 0.2 ml of a cDNA library on a single plate; the library has a titer of

Question

We plate 0.2 ml of a cDNA library on a single plate; the library has a titer of 2 x 106 bacteria per ml. We grow the plate over night to make a bacterial lawn and blot the plate against a nylon filter. We then hybridize (hyb) a probe of our favorite gene to the nylon filter, wash off the probe, and use x-ray film to see if anything hybridized. We find a spot that where the probe hybridizes, yeah! Now what? We then:

sequence the positive hybridization spot on the nylon using the Sanger technique.

go back to the plate and steak out the positive spot.

isolate DNA from the positive spot and send it out to be sequenced.

dilute the positive spot on the initial plate, and repeat the steps above.

BLAST the positive region of the lawn to narrow down our search.

sequence the positive hybridization spot on the nylon using the Sanger technique.

go back to the plate and steak out the positive spot.

isolate DNA from the positive spot and send it out to be sequenced.

dilute the positive spot on the initial plate, and repeat the steps above.

BLAST the positive region of the lawn to narrow down our search.

Explanation / Answer

since we got a bacterial lawn in which it is very difficult to point out a single colony. so we did hybridization and choose a specific region where probe bind. now in next step we dilute the positive spot on initial plate and again we repeat the steps above to find out the colony containing desired sequence.

so option 4.

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