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KAI-OLAF NETZER
b. Quickly chill on ice, add 2.5J..Llloading buffer, and load on to a formaldehyde/agarose gel prepared as follows:
Agarose
lOx MOPS buffer
Water
1.0 g
10.0 ml
73.0 ml
c. Heat to 100°C to dissolve the agarose, then cool to 50°C in a water bath.
Add 17 ml formaldehyde, mix and pour the gel immediately. W ork under
a fume hood and wear gloves. U se an electrophoresis chamber that is
reserved for RNA electrophoresis.
Northern blots 1. While the gel is still running, cut a sheet of fllter membrane to the size of
the gel. Soak in 20x SSC.
2. Set up the transfer apparatus as shown in Figure 1. Fill a traywith blotting
buffer (20x SSC). Make a platform above the buffer level, e.g. by inverting
a gel tray and cover it with a wick made from 3 sheets of fllter paper
(Whatman 3MM) saturated with 20x SSC. The wick should hang into
the buffer on both sides of the platform. Use a 10 ml glass pipet to
roll out air bubbles and to flatten the paper wick.
3. Po ur several ml of 20x SSC on top of the paper wick, then place the gel
without further treatment upside down on the wick. Carefully remove
any air bubbles trapped between gel and paper wick. Surround the
gel with cling film or plastic wrap to prevent buffer flow around the gel.
Note: The use of a glass pipet may be helpful toroll out air bubbles. Take care
not to distort or squeeze the gel.
4. Cover the gel with several ml of 20x SSC. Place the fllter membrane on top
of the gel. Make sure that no air bubbles are trapped between the gel and
the fllter. Mark the side of the fllter membrane that faces the gel with a
soft pencil.
5. Place 3 sheets of 3MM paper cut to size and pre-wetted with 20x SSC on
top of the fllter membrane.
6. Place a stack of dry absorbent paper (blotting paper, or paper towels) on
top of the 3MM paper (approximately 5-8 cm high).
7. Place a glass plate on top of the stack of paper, and put a 0.75-1 kg weight
on top.
KAI-OLAF NETZER
b. Quickly chill on ice, add 2.5J..Llloading buffer, and load on to a formaldehyde/agarose gel prepared as follows:
Agarose
lOx MOPS buffer
Water
1.0 g
10.0 ml
73.0 ml
c. Heat to 100°C to dissolve the agarose, then cool to 50°C in a water bath.
Add 17 ml formaldehyde, mix and pour the gel immediately. W ork under
a fume hood and wear gloves. U se an electrophoresis chamber that is
reserved for RNA electrophoresis.
Northern blots 1. While the gel is still running, cut a sheet of fllter membrane to the size of
the gel. Soak in 20x SSC.
2. Set up the transfer apparatus as shown in Figure 1. Fill a traywith blotting
buffer (20x SSC). Make a platform above the buffer level, e.g. by inverting
a gel tray and cover it with a wick made from 3 sheets of fllter paper
(Whatman 3MM) saturated with 20x SSC. The wick should hang into
the buffer on both sides of the platform. Use a 10 ml glass pipet to
roll out air bubbles and to flatten the paper wick.
3. Po ur several ml of 20x SSC on top of the paper wick, then place the gel
without further treatment upside down on the wick. Carefully remove
any air bubbles trapped between gel and paper wick. Surround the
gel with cling film or plastic wrap to prevent buffer flow around the gel.
Note: The use of a glass pipet may be helpful toroll out air bubbles. Take care
not to distort or squeeze the gel.
4. Cover the gel with several ml of 20x SSC. Place the fllter membrane on top
of the gel. Make sure that no air bubbles are trapped between the gel and
the fllter. Mark the side of the fllter membrane that faces the gel with a
soft pencil.
5. Place 3 sheets of 3MM paper cut to size and pre-wetted with 20x SSC on
top of the fllter membrane.
6. Place a stack of dry absorbent paper (blotting paper, or paper towels) on
top of the 3MM paper (approximately 5-8 cm high).
7. Place a glass plate on top of the stack of paper, and put a 0.75-1 kg weight
on top.