Two Hybrid Screen
Isolation and Purification of Sequencing Templates from Yeast Transformants



After obtaining yeast colonies from a two hybrid screen, typically the next step is to screen the plasmids in the transformants for sequences of interest. With several hundred transformants to check, this a labor intensive and time consuming process. Because the Merlin Core Services personnel have the process optimized and running on a daily basis, you can save significant time and expense. We will isolate plasmid from the yeast cells, transform, and amplify the plasmid in E. coli to produce a sequencing template.

Procedure:

  1. Grow yeast strain in selective media to maintain selection for plasmid.
  2. Isolate plasmid DNA from yeast using method as per pg 76
  3. Transform competent bacteria and select for the antibiotic resistance conferred by the plasmid isolated from the yeast.
  4. Plate on minimal bacterial media lacking trp or leu to select for desired plasmid (of the two involved in the two-hybrid system used) based on complementation of the yeast gene for bacterial nutritional deficiency.




Yeast Transformant Colony



Replica Plating Device


Two-Hybrid Characterization Screens

A. Isolation of DNA from Yeast
Catalog No.No. of PrepsPrice/prep
9906-1001-25$10.00
9906-10126-50$8.00
9906-10251-100$7.00
9906-103101-200$6.00
9906-104201-500$5.00
9906-105>500$4.50
A. Transformation of competent bacteria with DNA isolated from yeast.
9906-2001-25$8.00
9906-20126-50$7.00
9906-20251-100$6.00
9906-203101-200$5.00
9906-204201-500$4.00
9906-205>500$3.50
Includes replica plating on minimal media to select for plasmid of interest.
Preparation of sequencing template DNA from appropriate transformants.




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