16 Comparative Genomic Hybridization
205
- Phosphate Buffered Saline
- STY (0.25% trypsin)
- Methotrexate
- Thymidine
- Colcemid
Procedure
Metaphases from Mouse Embryonic Fibroblasts
1. Obtain two to six 12-day-old mouse embryos (C57 Black). If these need Day 1
to be dissected from the uterus, work in a sterile Petri dish using sterilized dissecting instruments and sterile media. Cultures should all be
prepared and maintained using sterile technique and a biological
safety hood.
2. Place embryos into a IS ml centrifuge tube. Add enough complete
medium to cover. Using the tip of a 10 ml pipette, smash the embryos
against the bottom ofthe tube repeatedly until a smooth homogenate is
obtained. Pass the homogenate through an 18-gauge needle several
times.
3. Transfer the homogenate to a sterile Petri dish and run the homogenate through the needle several more times.
4. Place I ml of the homogenate into a T-25 cell culture flask. Add 5 ml
complete medium to the flask and gently swirl. Cap flask(s) and place
horizontally in 37 DC 5% CO 2 incubator. Caps do not need to be loosened.
1. Examine cells for healthy attachment to flask using inverted phase con- Day 2
trast microscope.
2. Aspirate complete medium to remove dead and floating debris and
wash gently with 8 ml Hank's balanced salt solution, Ca++ and
Mg++ free.
3. Replace with 8 ml complete medium and return flask to incubator.
4. Repeat the Day 2 procedure daily until the cell layer is confluent.
205
- Phosphate Buffered Saline
- STY (0.25% trypsin)
- Methotrexate
- Thymidine
- Colcemid
Procedure
Metaphases from Mouse Embryonic Fibroblasts
1. Obtain two to six 12-day-old mouse embryos (C57 Black). If these need Day 1
to be dissected from the uterus, work in a sterile Petri dish using sterilized dissecting instruments and sterile media. Cultures should all be
prepared and maintained using sterile technique and a biological
safety hood.
2. Place embryos into a IS ml centrifuge tube. Add enough complete
medium to cover. Using the tip of a 10 ml pipette, smash the embryos
against the bottom ofthe tube repeatedly until a smooth homogenate is
obtained. Pass the homogenate through an 18-gauge needle several
times.
3. Transfer the homogenate to a sterile Petri dish and run the homogenate through the needle several more times.
4. Place I ml of the homogenate into a T-25 cell culture flask. Add 5 ml
complete medium to the flask and gently swirl. Cap flask(s) and place
horizontally in 37 DC 5% CO 2 incubator. Caps do not need to be loosened.
1. Examine cells for healthy attachment to flask using inverted phase con- Day 2
trast microscope.
2. Aspirate complete medium to remove dead and floating debris and
wash gently with 8 ml Hank's balanced salt solution, Ca++ and
Mg++ free.
3. Replace with 8 ml complete medium and return flask to incubator.
4. Repeat the Day 2 procedure daily until the cell layer is confluent.