Department of Ecology, Evolution and Organismal Biology
Iowa State University, Ames, IA 50011, USA
Note: This protocol is based on protocols from the Wurtele Lab & Chapter 7, Molecular Cloning, Third Ed.
<!>All steps involving formaldehyde or formamide should be carried out inside a hood.
<!> Formaldehyde causes Emphysema
<!> Do not use latex gloves with organic solvents - they will melt to your skin. Use nitrile.
Reagents and Buffers
27µl Formaldehyde (37% Solution), Normalized pH 7.0
150µl 10X MOPS buffer
571µl DEPC treated H2O
3µl 10mg/ml Ethidium Bromide
0.375mg Orange G
8µl Loading Buffer
2µl RNA Standard
10X MOPS Buffer
|0.4 M MOPS||41.86g||83.72g|
|100mM Sodium Acetate||6.805g (16.67ml 3M NaOAc)||13.61g|
|10mM EDTA||10ml 0.5 M EDTA||20.0ml 0.5 M EDTA|
Notes: All water used to make solutions must be DEPC treated. The EDTA solution must be normalized to pH 7.0 with Acetic Acid.
|44.1ml DEPC treated H2O||88.2ml||132.3ml||176.4ml|
|5.0ml 10X MOPS Buffer||10.0ml||15.0ml||20.0ml|
|0.9ml Formaldehyde (37% Solution)||1.8ml||2.7ml||3.6ml|
Notes: Mix all reagents except formaldehyde and microwave. Wait until the gel cools to 55 C then add formaldehyde and pour immediately. Gel will be slimy and fissile. Thin gels are better for transferring to membranes.
Formaldehyde Running Buffer
|882ml DEPC treated H2O||1.764L||3.528L|
|100ml 10X MOPS Buffer||200ml||400ml|
|18ml Formaldehyde (37% Solution)||36ml||72ml|
- All formaldehyde must be normalized to pH 7.0.
- You do not need to circulate the buffer in the rig because the concentration of formaldehyde is equilibrated.
- Run gel for 2-3 hours at 80V or, for finer resolution, run at 5V/cm for 4-5 hours.
Protocol updated May 2003. Many thanks to Ryan Rapp.