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.

载体描述：

pHISi-1 is a yeast integration and

reporter vector for use with the MATCHMAKER One-Hybrid System. pHISi-1 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. pHISi-1 was constructed

by transferring the HIS reporter gene from pHISi to the EcoR I/BamH I sites of pBR322. Leaky HIS3

expression in pHISi-1 is generally lower than that in pHISi, presumably due to differences in the

flanking vector sequence.

The yeast HIS3 gene is used as a selectable marker for integration into the nonfunctional his3 locus

of the YM4271 host strain after linearizing the vector at the Xho I or Afl II sites. 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. Because it does not carry the URA3 marker, pHISi-1 can be used (together

with pLacZi) to construct a dual HIS3/lacZ reporter strain. pHISi-1 cannot replicate autonomously

in yeast. pHISi-1 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.