13 Sequencing
203
20. Take the glass plates out of the chamber and remove the small plate
carefully so that the gel remains attached to the other plate; cut off
a small part of the left upper corner of the gel.
21. Place the gel on the plate in a suitable tray and fill with a solution containing lOo/o acetic acid/lOo/o methanol. Incubate for 15 min. This step
will wash out the urea from the gel.
22. Carefully remove the solution by aspiration, and rinse again with lOo/o
methanol for an additional 5 min.
23. Layer a piece of Whatman filter paper cut to size on to the gel. A void
entrapping air bubbles between the gel and the paper. The gelwill stick
to the paper. Invert the assembly, and position it on the edge of the lab
bench. Peel the paperdownward with gel attached, while carefully Iifting up the plate. Cut off one corner corresponding to the cut corner of
the gel. Cover the free gel side with plastic wrap and transfer to the vacuum gel drying apparatus.
24. Dry for 60 min at 80°C.
25. Remave the plastic wrap. In the darkroom place the gel in a cassette and
place a sheet ofX-ray film in direct contact with the gel. Bend one corner
ofthe film corresponding to the cut-off corner ofthe gel. This ensures
the correct alignment of autoradiogram and gel.
26. Expose overnight at room temperature.
Troubleshooting
There are many problems that may arise when performing sequencing reactions. Sameare due to improper reagents or DNA, others are due to formation of secondary DNA structure during DNA polymerization as well as
during electrophoresis. The following chapter willlist problems and their
possible underlying causes and will recommend changes of the protocol. It
may also help to adapt the technique to your specific needs.
• W eak signals or even blank films
- Did you follow all the steps or did you omit anything when performing
the sequencing reactions?
- Bad template DNA preparation? Problem should not occur using
commercial control DNA.
203
20. Take the glass plates out of the chamber and remove the small plate
carefully so that the gel remains attached to the other plate; cut off
a small part of the left upper corner of the gel.
21. Place the gel on the plate in a suitable tray and fill with a solution containing lOo/o acetic acid/lOo/o methanol. Incubate for 15 min. This step
will wash out the urea from the gel.
22. Carefully remove the solution by aspiration, and rinse again with lOo/o
methanol for an additional 5 min.
23. Layer a piece of Whatman filter paper cut to size on to the gel. A void
entrapping air bubbles between the gel and the paper. The gelwill stick
to the paper. Invert the assembly, and position it on the edge of the lab
bench. Peel the paperdownward with gel attached, while carefully Iifting up the plate. Cut off one corner corresponding to the cut corner of
the gel. Cover the free gel side with plastic wrap and transfer to the vacuum gel drying apparatus.
24. Dry for 60 min at 80°C.
25. Remave the plastic wrap. In the darkroom place the gel in a cassette and
place a sheet ofX-ray film in direct contact with the gel. Bend one corner
ofthe film corresponding to the cut-off corner ofthe gel. This ensures
the correct alignment of autoradiogram and gel.
26. Expose overnight at room temperature.
Troubleshooting
There are many problems that may arise when performing sequencing reactions. Sameare due to improper reagents or DNA, others are due to formation of secondary DNA structure during DNA polymerization as well as
during electrophoresis. The following chapter willlist problems and their
possible underlying causes and will recommend changes of the protocol. It
may also help to adapt the technique to your specific needs.
• W eak signals or even blank films
- Did you follow all the steps or did you omit anything when performing
the sequencing reactions?
- Bad template DNA preparation? Problem should not occur using
commercial control DNA.