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General guidelines for electrophoresis
1. Ultrapure or electrophoresis grade reagents should be used.
2. Sequencing gels should be made fresh. Store solutions no longer than one
week in the dark at 4°C. Commercial preparations of acrylamide gel mixes in
liquid or powder form (RapidGel gel mixes—see ‘Related Products’) should
be used according to manufacturers recommendations.
3. Gels should be prepared 2-20 hours prior to use, and pre-run for ~15
minutes.
4. When reading longer sequences, it is usually convenient to run gels
overnight with a timer-controlled power supply. Gel runs of 18-24 hours at
40-50 watts are often necessary for reading in the 400-600bp range.
5. Loading 8 adjacent lanes in a pattern that abuts all pairs of lanes (
e.g.
GATCGTAC) aids reading closely spaced bands.
6. Gels should be soaked in 5% acetic acid, 15% methanol to remove the urea.
Soaking time depends on gel thickness. Approximate minimum times are 5
minutes for 0.2mm gels, 15 minutes for 0.4mm gels and 60 minutes for field
gradient (0.4-1.2mm wedge) or formamide gels. Drying should be done at
moderate temperature (80°C) to preserve resolution.
7. If RapidGel-XL is used, the gel does not need to be soaked. In fact, soaking
RapidGel-XL gels will cause swelling thereby affecting band resolution in the
final result.
8. For
33
P gels, autoradiography must be done with direct contact between the
dried gel and the emulsion side of the film. Gels dried without prior soaking
(leaving plastic-wrap on helps to prevent the film from sticking to the
incompletely-dried gels) will require longer drying and exposure times but
give sufficient resolution for most purposes.
9. Good autoradiography film can improve image contrast and resolution. We
recommend Kodak Biomax™ MR or Hyperfilm™-bmax autoradiography film.
10.In general, overnight to 36 hour exposures are sufficient when using fast film
such as Hyperfilm™-MP.
11.The use of tapered spacers (‘wedge' gels) improves overall resolution and
allows more nucleotides to be read from a single loading (4).
TROUBLESHOOTING
Problem
Possible causes and solutions.
Extensions appear short (read length limited to less than 200 bases)
1. If using dITP, increase time of extension step in cycles to 5-10 minutes and
decrease temperature to 60°C. See figure 2.
2. Too much template DNA. In some cases, the use of too much DNA,
especially PCR product DNA, can exhaust the supply of ddNTPs. Use less