For selection of recombinants

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asked Dec 8, 2017 by sforrest072 (157,439 points) 61 411 949

For selection of recombinants, insertional inactivation of antibiotic marker has been superceded by insertional inactivation of a marker gene coding for a chormogenic substrate. Give reasons

1 Answer

+1 vote
answered Dec 8, 2017 by mdsamim (213,225 points) 5 10 15
selected Dec 8, 2017 by sforrest072
 
Best answer

Selection of recombinants due to inactivation of antibiotics is a laborious process as it requires:

 (i) a vector with two antibiotic resistance marker 

(ii) preparation of two kinds of media plate, with one antibiotic each

Transformed cells are first plated on that antibiotic plate which has not been insertionally inactivated (ampicillin) and incubated overnight for growth of transformants. For selection of recombinants, these transformants are Replica plated on second antibiotic (tetracycline) plate (which got inactivated due to insertion of gene). Non-Recombinants grow on both the plates (one carrying ampicillin and the other carrying tetracycline ) while recombinants will grow only on ampicillin plate.

This entire exercise is laborious and takes more time (two overnight incubation) as well. However, if we choose the second option (insertional inactivation of a marker that produces colour in the presence of a chromogenic compound), we can distinguish between the recombinants and non-recombinants on a single medium plate (containing one antibiotic and the chromogenic compound) after overnight growth. Hence I would choose a marker which produces a coloured compound but gets inactivated due to insertion of foreign DNA. 

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