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324 Thibodeau-Beganny and JoungTypically, DNA is eluted in 50 µL 0.1X EB (a 1X EB stock is provided in theQIAgen kit).9. To verify uptake of the annealed oligonucleotides in the pKJ1712 plasmid, 5 µL ofeach candidate DNA is digested with EcoRI and HindIII. As a control, plasmidpKJ1712 is also digested with EcoRI and HindIII. Recombinants that have taken upthe annealed oligonucleotide insert should yield five bands of sizes 6109, 1006, 963,431, and 190 bp. By contrast, pKJ1712 should yield five bands of sizes 6109, 1006,963, 456, and 190 bp (see Note 3).10. Plasmids that look correct by restriction digest should be sequenced to confirm thetarget DNA-binding site. Primer OK.181, a primer which anneals to the senseDNA strand just downstream of (and pointing back toward) the target binding site,to verify the sequence of the insert is used (see Note 4).3.1.2. F′ Episome Recombination and Transfer3.1.2.1. RECOMBINATION OF REPORTER PLASMID SEQUENCES ONTOTHE F′ OF STRAIN CSH100As shown in Fig. 4, the reporter plasmid contains portions of the lacI q andlacZ gene that are also present in the F′ found in strain CSH100. These regionsof matching sequence can serve as point of recombination between the reporterplasmid and the F′. A double crossover event at both regions of sequence identitycan lead to transfer of a portion of the reporter plasmid onto the F′.1. Streak out F − strain KJ1C on a LB/Tet plate and grow at 37°C overnight.2. The next day, transform F + strain CSH100 with the reporter plasmid and plateenough to obtain a confluent lawn of transformants on LB/Kan plate. Incubateovernight at 37°C.3.1.2.2. TRANSFER OF F′S FROM CSH100 TO KJ1C BY BACTERIAL MATINGThe population of transformed CSH100 cells will contain a small numberof cells in which a single recombination event has led to integration of thereporter onto the F′ (Fig. 5). In an even smaller number of cells, a doublerecombination event will have exchanged the target DNA-binding site and promoterpresent on the reporter plasmid with the promoter on the F′ (Fig. 5). Asis described in this step, all F′s (recombinant and nonrecombinant) in theCSH100 transformants are transferred to the F − strain KJ1C by mating. In asubsequent step, the desired double recombinant F′ that have been transferredto strain KJ1C can be identified by plating on appropriate selective medium(Fig. 5 and see Step 9).1. Scrape the confluent plate of CSH100 transformants with a sterile wooden stickand transfer cell paste to a sterile 25-mm glass tube containing 10 mL of LB(see Note 5).