Add 73.3 ml 2x distilled water into a 250 ml Erlenmeyer flask.
Add 1.5, 1.8 or 2 g of agarose.
Bring to a boil in the microwave.
Go to the hood and add:
16.7 ml formaldehyde
10 ml 10x MAE.
Pour into the mold.
Wait 30’ and transfer into the tank having 1liter 1x MAE.
Rinse the wells just before adding the samples.
Run for 3 – 5 hrs at 75 – 80 mA.
Samples are in:
50% formamide
2.2 M formaldehyde
1 x MAE
Put at 65oC for 5’
Quick cool
Add loading dye.
Do not store gel before the run – make it and run.
Do not stain gel to be blotted.
Rinse gel to be blotted in 2x distilled water for a few seconds.
Blot onto BA-83, BA-85 or Nytran, using 10x SSC.
Bake for 2 hrs at 80oC when using BA-83 or 85, and UV-cross link with Nytran.
10x MAE buffer
200 mM MOPS
50 mM Na Acetate
10 mM EDTA pH 7.0
Note: Do not autoclave the buffer.