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dtmc

Houston, Texas

BCM is a research-intensive medical center dedicated to a rapid pace of discovery and its equally rapid translation to patient care.
Mouse Embryonic Stem Cell Core Facility
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Clone Expansion

Requirements:

Investigators must receive approval from the director of the core for a project to be initiated.

Cell lines submitted to the core must be tested for the presence of murine virus. Please, refer to Murine Virus Testing page for instructions.

Complete and submit the Clone Expansion Request Form. Please, be very specific when describing well location. Refer to Pricing for pricing information.

Submit cells as frozen stock in cryovials or 96 well plates.

Bring cells to the core at scheduled times or project may be delayed.

Prior to scheduling any injections or electroporations using expanded clones, investigators must make DNA from each clone, after receiving cells from the core, in order to confirm the correct cells were expanded.

Service:

The core will bring cells into culture on Fridays.

96 Well Clone Expansions:

One well of a 96 well plate will be expanded up to one, 100mm plate. This expansion is done in gradual step by step form; taking cells from one well of a 96 well plate into one well of a 24 well plate, into one, 60mm plate and finally into one, 100mm plate. From the 100mm plate, cells will be frozen down according to the Cryopreservation of Mouse ES Cells protocol. At freezing, an aliquot of cells will be plated in a feeder free, 60mm plate which will be turned over to the investigator for re-confirmation of clones by mini southern analysis.

This process should yield two to three vials of frozen stock from each clone. The investigator may choose for the core to keep this frozen stock in LN2 storage indefinitely or may keep cells in his/her lab LN2 storage.

Expansion from Single Cryovial:

If an investigator has a single cryovial of mouse embryonic stem cells that he/she would like to expand, the core will be available to provide this service.

The vial will be thawed and placed in one 60mm dish, previously coated with feeder cells. After the cells reach approximately 80% confluency cells will be expanded into one or two, 100mm plates for freezing. The expansion into one or two plates will depend on the number of vials the investigator would like.

This process should yield three to five vials of frozen stock from each original vial. At the investigator's request, the core will hold a total of three vials for each line in LN2 storage indefinitely. However, other arrangements for LN2 storage must be made for additional vials.

Expected Timeline:

After contacting the director of the core for project approval, investigators must complete and submit the Clone Expansion Request Form. The core supervisor will e-mail the contact person within three working days after the form is submitted to inform the lab of the scheduled date to bring cells to the core. Every request will be taken on a first come, first serve basis.

96 Well Clone Expansions:

Timeline will depend on how well the cells recover from the freezing process. It will require approximately 14 days in culture, to bring cells from one well of a 96 well plate to one 100mm plate. It will take an additional 4 to 5 days to get feeder free plate with cells for DNA extraction.

Therefore, investigators can expect to be contacted by core personnel to come pick up cells for DNA, approximately 20 days after cells are thawed from a 96 well plate.

Expansion from Single Cryovial:

Timeline will depend on how well the cells recover from the freezing process. It will require approximately four days in culture, to bring cells from one 60mm plate to one 100mm plate. It will take an additional 4 to 5 days to get feeder free plate with cells for DNA extraction.

Therefore, investigators can expect to be contacted by core personnel to come pick up cells for DNA, approximately 10 days after cells are thawed.

Problems:

It is highly recommended that investigators keep backup vials of cells submitted to the core. The core cannot guarantee the recovery in culture, of cell lines frozen down by other groups.

In the event that cells do not grow well in culture, the investigator will be notified as soon as core personnel become aware of the problem so that additional vials may be submitted by the investigator.

The director of the core will deal with problems on a case by case basis.

After service agreement:

We would appreciate that any publications arising from the generation of cell lines by this core carry an appropriate acknowledgment of this service.

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