[Pombelist] Question regarding thawing Bioneer library
I am planning to start working with version 5 of the haploid Pombe deletion library from Bioneer and was wondering if anyone could provide a protocol of how to best bring up the strains from frozen glycerol stocks?
The library is a fresh copy from bioneer and I tried pinning directly onto YES agar + G418 using a Singer rotor HDA. Unfortunately, many of the strains did not grow (approximately 10%). I have been suggested to first pin to YES plates without selection before replicating onto YES + G418 for selection, but thought it would be best to ask here first.
-
Official comment
This is a common problem. The plates have round bottom wells and cells are mostly sedimented. What helps is:
1. Set the rotor to 2d source mix without distance clearance between pin and bottom of well. Repeat pinning 3 times. I normally leave plates to dry and re-pin.
2. Using a plate incubator/shaker gently twirl the plates so cells detach from bottom. Do 3d source mix and do repeated pinning.
3. In case the results are very poor, work with batches of 3-4 plates, using a multipipette very gently bring the cells in solution. Then do 3d source mix and repeated pinning.
I always use YES+G418.
Please sign in to leave a comment.
Comments
1 comment