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332 Thibodeau-Beganny and Joung2. Prepare miniprep plasmid DNA from the saturated 4 mL overnight cultures usingQIAgen’s QIAprep Spin Miniprep Kit (cat. no. 27106) and their protocol with thefollowing differences:a. Perform triple washes with both PB and PE buffers (see Note 12).b. Elute the DNA with 60 µL of prewarmed (60°C) 0.1X EB (see Note 13).3. Send the plasmids for sequencing with sequencing primer OK.61, a sense strandprimer, which anneals just upstream of the region encoding the zinc fingerdomains.4. Notes1. It is important to dissolve the amino acids in each of the six solutions in preciselythe order listed as this avoids potential solubility issues. Typically, the amino acidmixture is kept for no more than 2–3 mo.2. 3-AT should be prepared using gloves. In addition, it has been found that thesolubility and purity of 3-AT varies from lot to lot. Some preparations have theappearance of a white powder whereas others look like brown flakes. For certainlots, it has been found that heating the solution to 50°C can aid with solubility.3. These digests are run on 5% polyacrylamide gels made with 0.5X TBE buffer tovisualize the relatively small change in fragment size in clones that have taken upthe annealed oligonucleotide insert.4. The entire sequence between the unique EcoRI site (positioned just upstream ofthe zinc finger domain-binding site) and the unique SalI site (positioned at thestart site of transcription) will be sequence verified. Verifying this entire span ofsequence ensures that both the zinc finger-binding site and the promoter do nothave undesired mutations.5. It has been found that using a resuspension of multiple transformed CSH100colonies rather than an overnight culture grown from a single transformed colonyhelps ensure that a relatively consistent percent of transformed CSH100 cellscontain the desired double-recombinant F′.6. Set vortex to half-maximum speed to ensure that resuspension does not spill overthe top of the glass tube.7. The initial density of this subculture should correspond to OD 600of approx 0.1(i.e., prelogarithmic phase). Depending on the density of the resuspension culture,more or less culture will be added as needed, to achieve this target OD 600.8. Occasionally, some small colonies are observed on the LB/TKS plates. Pickingthese colonies is avoided because it has been found that these colonies do not yieldthe desired recombinants.9. KJ1C cells will not grow unless proline and histidine are provided in their mediaowing to deletion of the proAB gene cluster and a deletion within the hisB gene,respectively. A double recombinant F′ transferred from CSH100 cells harbors anintact proAB gene cluster and expresses a low level of the yeast HIS3 gene, whichis sufficient to complement the hisB deletion of strain KJ1C. Thus, KJ1C cells thatreceive a double recombinant F′ should be able to grow on M9 minimal mediumlacking proline and histidine.