Yeast Cell Banks

Suggested Testing for Certification and Qualification of Yeast for Master Cell and Working Cell Banks
Culture Identity

• Cellular morphology to be determined by the Gram stain
• Genetic identity will be determined by  PCR analysis & sequencing of  25-28S rRNA

Viability determination by CFU assay/colony counting
Purity - plating on complex media (determination of purity in a microbial culture using differential agars), Gram stain
Genetic Stability

• Determination of percentage of cells retaining expression construct. (Retention of the recombinant construct).  Replica plating on selective and non-selective media.
• Plasmid or insertion copy number.  Determination of the number of copies of construct in the host genome or episomal copies/cell if not inserted. Analysis of gene copy number by QPCR amplification of specific nucleic acids and fluorescent probe technology.
• Structural integrity of the HB surface antigen gene to be assessed by Southern blot analysis if the gene of interest is integrated into genomic DNA.  Not required if the construct is episomal.

Sequence integrity of the gene of interest: 

• DNA sequencing using fluorescent dye terminators of PCR – amplified products prepared from cDNA.  Sequencing: Determination of the sequence of the gene of interest to verify the consistency of the sequence over multiple generation

Submission Forms
Microbial-Yeast Cell Bank Characterization Form (ZZ)