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Benjamin Costantino 5/05/05 Northern Blot with DIG
 

Summary: Benjamin Costantino 5/05/05
Northern Blot with DIG
Labeling
1. DNA template (10ng 30ug) to 15ul and boil 100 C for 10 min.
2. Snap cool on ice for 5 min.
3. Transfer: (DIG-DNA Labeling Kit: Roche #11 175 033 910)
a. 2ul 10x Hexanucleic mix (#5)
b. 2ul dNTP Label mix (#6)
c. 1ul Klenow (#7)
4. Total of 20ul, mix
5. Place tube in 37C up to overnight
6. Kill Klenow by heating 65C for 10 min.
7. Add up to 200ul; Spin purify with G50 Sephadex 2min. @ 1000rpm; collect in
screw cap tube.
Hybridization
1. Preheat EasyHyb ~ 40mls at 50C
2. Gently agitate blot with ~30mls preheated EasyHyb for 30min. (minimum)
3. Denature DIG-DNA probe and salmon sperm DNA (final conc. 100ug/ml) - boil
10min. and then Snap cool on ice for 5min.
4. Add DIG-DNA probe and salmon sperm DNA to 5mls of preheated EasyHyb.

  

Source: Andres, Andrew - School of Life Sciences, University of Nevada at Las Vegas

 

Collections: Biology and Medicine