RNA Gels With Formaldehyde Electrophoresis

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
                                                                                                                                                                           

Protocol updated May 2003. Many thanks to Ryan Rapp.