Cell biology

Reagents:         Alexa Fluor 488 Mouse Anti-BrdU (1:8 in stain buffer)

                        BrdU (16 µL per 5 mL of media)

Fletcher Lab DNA Extraction Protocol



A. Sample Preparation (before Proteinase K Digestion)



A1. Frozen Tumor Processing


a) Using scalpels, cut off small piece of tumor into a Petri dish on ice

-Avoid bloody and yellow (fatty or necrotic) parts

-note: we routinely use this protocol for DNA isolations from very small (1x1x1mm) tumor chunks - expected yield of genomic DNA is ~2 µg.

Note: volumes in red are appropriate for round petri dish cultures--feel free to add other volumes for different plate or flask types in another color.

See also: Counting hES cells using nucleocounter


1. Warm media in hood, 0.25% Trypsin in 37^oC waterbath.

2. Make Trypsin inhibitor solution:

Label a 50ml Falcon tube ‘TI’

How to plate cells onto chamber slides for immunocytochemistry, staining, etc.

1. Prepare culture plate (or flask) by covering bottom with a thin layer of 0.1% gelatin.

2. Incubate gelatin in plate for at least 1.5 hrs, or ON.

3. Aspirate media.