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KAI-OLAF NETZER
10. Po ur several ml of 20x SSC on top of the paper wick, then place the gel
upside down on the wick. Carefully remove any air bubbles trapped in
between the 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.
11. 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.
12. Place 3 sheets of3MM paper cut to size and pre-wetted with 20x SSC on
top of the filter membrane.
13. Place a stack of dry absorbent paper (blotting paper, or paper towels) on
top of the 3MM paper (approximately 5-8 cm high).
14. Place aglass plate on top ofthestackofpaper, and puta0.75-1 kgweight
on top.
15. Allow transfer of DNA to proceed for 12-16 hours (e.g., overnight).
Make sure that there is enough 20x SSC in the reservoir (1-3 1).
16. After completion of capillary transfer, carefully disassemble the blotting apparatus. Transfer the membrane together with the gel to a dry
filter paper, gel side up. Mark the position of the gel and the wells on the
fllter membrane by using a soft pencil. Peel off the gel.
Note: To check efficiency of transfer, the gel may be stained with ethidium
bromide ( 1 J..Lg/ml) in water for 20 minutes, and then photographed on a UV
transilluminator.
17. Wash the filter membrane briefly in Sx SSC to remove any traces of
agarose.
18. Place the filter membrane briefly on a dry sheet of 3MM paper to dry.
19. Fix the DNA to the fllter membrane. For nitrocellulose, hake for 2 hours
at 80°C in a vacuum oven. For nylon, expose the filter DNA side down to
UV light for 3-5 minutes (for optimizing DNA fixation, see below).
Note: Optimizing DNA fixation to the fllter by UV crosslinking may increase
the signal strength upon hybridization. The optimal UV irradiation time
may be determined experimentally: run an agarose gel with 5 identicallanes
of control DNA suchthat each lane is separated from the next by an empty
lane. Treat and blot the gel as described above, and expose each lane to UV
KAI-OLAF NETZER
10. Po ur several ml of 20x SSC on top of the paper wick, then place the gel
upside down on the wick. Carefully remove any air bubbles trapped in
between the 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.
11. 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.
12. Place 3 sheets of3MM paper cut to size and pre-wetted with 20x SSC on
top of the filter membrane.
13. Place a stack of dry absorbent paper (blotting paper, or paper towels) on
top of the 3MM paper (approximately 5-8 cm high).
14. Place aglass plate on top ofthestackofpaper, and puta0.75-1 kgweight
on top.
15. Allow transfer of DNA to proceed for 12-16 hours (e.g., overnight).
Make sure that there is enough 20x SSC in the reservoir (1-3 1).
16. After completion of capillary transfer, carefully disassemble the blotting apparatus. Transfer the membrane together with the gel to a dry
filter paper, gel side up. Mark the position of the gel and the wells on the
fllter membrane by using a soft pencil. Peel off the gel.
Note: To check efficiency of transfer, the gel may be stained with ethidium
bromide ( 1 J..Lg/ml) in water for 20 minutes, and then photographed on a UV
transilluminator.
17. Wash the filter membrane briefly in Sx SSC to remove any traces of
agarose.
18. Place the filter membrane briefly on a dry sheet of 3MM paper to dry.
19. Fix the DNA to the fllter membrane. For nitrocellulose, hake for 2 hours
at 80°C in a vacuum oven. For nylon, expose the filter DNA side down to
UV light for 3-5 minutes (for optimizing DNA fixation, see below).
Note: Optimizing DNA fixation to the fllter by UV crosslinking may increase
the signal strength upon hybridization. The optimal UV irradiation time
may be determined experimentally: run an agarose gel with 5 identicallanes
of control DNA suchthat each lane is separated from the next by an empty
lane. Treat and blot the gel as described above, and expose each lane to UV