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1. Remove and discard culture medium. 
2. Briefly rinse the cell layer with Ca++/Mg++ free Dulbecco's phosphate-buffered saline (D-PBS) or 0.05% (w/v) Trypsin - 0.53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
3. Add 1.0 to 2.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes). Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal. 
4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting. 
5. Add appropriate aliquots of the cell suspension to new culture vessels. Incubate cultures at 37°C.

Subcultivation ratio: A subcultivation ratio of 1:3 to 1:8 is recommended.
Medium renewal: Every 2 to 3 days

CSF1PO: 11,12
D13S317: 12,14
D16S539: 9,13
D5S818: 8,9
D7S820: 11
TH01: 7, 9.3
TPOX: 11
vWA: 16,19
Amelogenin: X

DuBridge RB, et al. Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system. Mol. Cell Biol. 7: 379-387, 1987. PubMed: 3031469

Pear WS, et al. Production of high-titer helper-free retroviruses by transient transfection. Proc. Natl. Acad. Sci. USA. 90: 8392-8396, 1993. PubMed: 7690960