Wendel Lab
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
Loading Buffer
750µl Formamide
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
RNA Markers
8µl Loading Buffer
2µl RNA Standard
10X MOPS Buffer
| Absolute | 500ml | 1L |
| 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.
Formaldehyde Gel
| 50ml | 100ml | 150ml | 200ml |
| 0.5g Agarose | 1.0g | 1.5g | 2.0g |
| 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
| 1L | 2L | 4L |
| 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.