Berglund:Denaturing Agarose

From OpenWetWare
Jump to navigationJump to search

RNA Denaturing Gel

total volume =30ml

Heat in microwave:
15ml nanopure water
0.3g agarose (VWR IB70042)

Make seperately and then add to cooled gel:
3ml 10x MOPS
5ml Formaldehyde
5ml nanopure water

1. running buffer is made: 50ml 10x MOPS + 450ml nanopure water
2. loading dye is made with EtBr at a final concentration of 0.13µg/µl (6.5µl @10µg/µl in 500µl of RNA loading dye)
3. Gel is poured into an Rnase-away cleaned gel box (minimum volume is 20ml gel) and allowed to polymerize for 30 minutes. The approximate thickness of the gel is 3mm.
4. Load 1.3µl of RNA (ladder or transcription product or mRNA) + 2µl of loading dye/EtBr + 1µl promega dye + 6µl of 1xMOPS
5. Heat to 95° step number 4 for 2 minutes
6. Spin down and then load all of it onto the gel.