Chromosomal integration of the pDUAL plasmid into S.pombe

Protocol taken from the paper by Akihisa Matsuyama, Atsuko Shirai et al. from 2004 “pDUAL, a multipurpose, multi copy vector capable of chromosomal integration in fission yeast” Yeast 2004; 21: 1289-1305

pDUAL sequences can be found here

Contents

1. Procedure
2. Notes

1. Procedure

1. The pDUAL plasmid is digested by restriction enzyme NotI (compatible with Cutsmart buffer) releasing the 3kb ars1-ura4+ fragment and leaving a linearized plasmid which has part of the fission yeast leucine gene on either end.
2. If possible, the ars1-ura4+ fragment may be further digested using restriction enzymes found only in this DNA segment.
3. At this point a gel extraction can be performed to isolate the linearized plasmid from the 3kb fragment and undigested plasmid, however I have performed successful integrations without following this step.
4. If not doing a gel extraction, simply run the sample through the PCR clean up kit to remove all enzymes.
5. The linearized pDUAL can be transformed into competent cells that are leucine auxotroph by the quick lithium acetate method. Integration occurs by homologous recombination between the leucine gene fragments on pDUAL and the endogenous leucine locus. The recombination creates a functioning leucine gene.
6. The resulting integrated strains are selected on leucine drop-out plates.

2. Notes

Non digested pDUAL may be maintained extra-chromosomally in S.pombe by selection on uracil drop-out plates

Note: Strains with an integrated pDUAL plasmid must be kept in/on media without leucine to maintain the selection. If this is not done the 2 leucine sites created by integration can recombine causing the loss of the pDUAL segment of DNA. We have seen this happen with integrants grown in liquid YES media.