User:Torsten Waldminghaus/Notebook/EMSA SeqA

Gel

15% PAA gel to seperate biotinilated from unbiotinilated strand after methylation:

15% Acrylamid/Bisacrylamid/Urea solution

for 250 ml:

• in glas mix:
• 93.75 ml Acrylamid/Bisacrylamid-Solution (40%, Verhältnis 19:1, z.B. Rothiphorese Gel 40 von Roth)

• 105 g Urea
• add A. dest to ca. 200 ml and stirring under light heating (cover glass)
• +25 ml 10x TBE
• fill up to 250 ml with A. dest
• store at 4 °C in dark flask
• (filtering or degasing is not required)

To use for a gel add about 400 μL 10% APS for 100ml gel solution and 100 μL TEMED.

Gel schön lange bei 20 mA (ca. 1000 V) vorlaufen lassen (min. 30 Min), zum Temperieren. Gel bei 25 mA (ca. 1200 V) laufen lassen, bis Bromphenol-Blau unten raus gelaufen ist (bei 20 cm ca. 1-2h).