Lets say that we are interested in the E. coli genes that are involved in synthesis of histidine. To find insertion mutants that can not synthesize histidine(His"we could screen amongst our collection of 2x10- random Tn5 insertions to find those that are His The easiest way to do this would be to plate out the collection of insertions at a density of 200 colonies per plate(100 plates total). Each of these master plates would then be replica plated (first by transfer to a sterile piece of velvet) to a plate that contains histidine and also to a plate that lacks histidine. His insertion mutants would be identi fied as colonies that can not grow on the plates that lack histidine. Note that the same collection of random Tn5 insertions can be screened multiple times to find interesting mutations with different phenotypes 3)Identify His- Tn5 insertion mutants by replica plating to find colonies that specifically can not grow on plates that dont contain histidine Once we have a set of His" insertion mutations (in the present example, one might expect to find 10-20 different His" mutants), the affected gene(s) can be identified by the simple fact that they will be "tagged"by the inserted Tn5 sequences. The easiest way identify the site of insertion is by performing a special PCR amplification of the dNa fragment that corresponds to the novel junction betweenTn5 and the bacterial chromo somal sequences. Ordinarily PCR reactions are carried out using two DNA primers, each of which corresponding to an end of the sequence to be amplified. When we want to amplify a junction fragment we can use as one of the primers a sequence that lies near the end of Tns but we won't yet know the relevant chromosomal sequence to allow the other primer to be designed. There are several tricks that can be used to circumvent this problem, which are too complicated to describe here. Suffice it to say that there are ways that the junction fragment can be amplified by pCr using only sequences defined by the Tn5 portion of the junction fragment 4)Use the known sequence of the end of Tns to PCr amplify a fragment that spans the junction between the end of Tn5 and the E coli chromosomal site that was the target for insertion DNA sequencing of the amplified junction fragments will give the identity of the target sequences. Since we know the dNa sequence of the entire E coli chromosome, the gene that was the target for Tn5 insertion can be identified unambiguously 5)The dNa sequence of the junction fragments will identify all of the genes that have been inactivated to give the His" phenotypeLet’s say that we are interested in the E. coli genes that are involved in synthesis of histidine. To find insertion mutants that can not synthesize histidine (His–) we could screen amongst our collection of 2x104 random Tn5 insertions to find those that are His–. The easiest way to do this would be to plate out the collection of insertions at a density of 200 colonies per plate (100 plates total). Each of these master plates would then be replica plated (first by transfer to a sterile piece of velvet) to a plate that contains histidine and also to a plate that lacks histidine. His– insertion mutants would be identi￾fied as colonies that can not grow on the plates that lack histidine. Note that the same collection of random Tn5 insertions can be screened multiple times to find interesting mutations with different phenotypes. 3) Identify His– Tn5 insertion mutants by replica plating to find colonies that specifically can not grow on plates that don’t contain histidine. Once we have a set of His– insertion mutations (in the present example, one might expect to find 10-20 different His– mutants), the affected gene(s) can be identified by the simple fact that they will be “tagged” by the inserted Tn5 sequences. The easiest way to identify the site of insertion is by performing a special PCR amplification of the DNA fragment that corresponds to the novel junction betweenTn5 and the bacterial chromo￾somal sequences. Ordinarily PCR reactions are carried out using two DNA primers, each of which corresponding to an end of the sequence to be amplified. When we want to amplify a junction fragment we can use as one of the primers a sequence that lies near the end of Tn5 but we won’t yet know the relevant chromosomal sequence to allow the other primer to be designed. There are several tricks that can be used to circumvent this problem, which are too complicated to describe here. Suffice it to say that there are ways that the junction fragment can be amplified by PCR using only sequences defined by the Tn5 portion of the junction fragment. 4) Use the known sequence of the end of Tn5 to PCR amplify a fragment that spans the junction between the end of Tn5 and the E. coli chromosomal site that was the target for insertion. DNA sequencing of the amplified junction fragments will give the identity of the target sequences. Since we know the DNA sequence of the entire E. coli chromosome, the gene that was the target for Tn5 insertion can be identified unambiguously. 5) The DNA sequence of the junction fragments will identify all of the genes that have been inactivated to give the His– phenotype