Product Data Sheet

Recovery

1. Obtain an LB agar plate with the appropriate antibiotic.
2. Using a sterile pipette tip, touch the bacteria growing within the punctured area of the stab culture. (A sterilized wire loop or sterile toothpick can be used in place of a sterile pipette tip.)
3. Run this tip lightly over a section of the plate, as shown in the figure, to create streak #1.
4. Using another sterile pipette tip, pass through streak #1 and spread the bacteria over a second section of the plate, to create streak #2.
5. Using a third sterile pipette tip, pass through streak #2 and spread the bacteria over the last section of the plate, to create streak #3.
6. Grow overnight in a 37 ^o C incubator (unless a different growth temperature is indicated on the plasmid datasheet).
7. In the morning, single colonies should be visible. If the bacterial growth is too dense, re-streak onto a new agar plate to obtain single colonies.

载体信息：

载体描述：

pHISi is a yeast integration and reporter vector for use with the MATCHMAKER One-Hybrid System. pHISi contains the yeast HIS3 gene downstream of the MCS and the minimal promoter of the HIS3 locus (PminHIS). Cis-acting sequences of interest (i.e., target elements) can be inserted into the MCS. Without activation by a target element, constitutive HIS3 expression from PminHIS is very low in yeast, but allows enough growth to select for integration when constructing HIS3 reporter strains. During library screening, the leaky expression of HIS3 is controlled by adding 3-AT to the medium.

The yeast URA3 and HIS3 genes of pHISi can be used as selectable markers for integration into the nonfunctional ura3 and his3 loci, respectively, of the YM4271 host strain. Before integrating, the vector is linearized at the Xho I or Afl II sites (his3 locus) or at the Apa I site (ura3 locus). The Kpn I site cannot be used for integration because it cuts within the coding region of the HIS3 gene, and that region is deleted in YM4271. pHISi cannot replicate autonomously in yeast. The plasmid contains a bacterial Col E1 origin (ori) and the ampicillin resistance gene (Ampr) for propagation and selection in E. coli. Unique restriction sites are in bold.