13 Sequencing
205
ever, you should be aware, that diTP destabilizes DNA lowering the
yield. Formamide, added to the polyacrylamide solution, should also
prevent fragments from forming secondary structures during electrophoresis.
• Bands at the same position in 2 or 3 lanes throughout the gel
- Spontaneous deletions may occur during phage growth leading to 2
different template DNAs. Limit the time allowed for growth to 6 hours.
• Faint bands at the bottom of the gel
- If you are primarily interested in the first 50 bp adjacent to the primer,
dilute the dNTP-concentration during the labeling reaction by a third,
increase the ddNTP-concentration by 3-fold, increase the amount of
template and restriet the time allowed for labeling and termination to
1-2 min. Use a short electrophoresis run (1-2 hours).
• Faint bands at the top of the gel
- Increase the concentration of dNTPs by 2-fold and reduce the concentration of ddNTPs by a half during the termination reaction.
• Faint bands all over the gel
- Increase the amount of template and reduce the time for termination
to 1 min. If too long a time is chosen, extraneous nuclease activity may
cause degradation of the newly synthesized DNA strand.
References
Ausubel R, Brent R, Kingston RE, Moore DD, Seidman JG, SmithJA, Struhl K (eds) (1990)
Current protocols in molecular biology, Volumes 1 and 2. Greene Publishing Associates and Wiley-Interscience
Barnes WM, Bevan M, Son PH (1983) Kilo-sequencing: creation of an ordered nest of
asymmetric deletions across a large target sequence carried on phage M13. Methods
Enzymol101:98
Biggin MD, Gibson TJ, Hong GF (1983) Buffer gradient gels and 35 S labelas an aid to
rapid DNA sequence determination. Proc Natl Acad Sei U.S.A. 80:3963
Church GM, Kieffer-Higgins S (1988) Multiplex DNA Sequencing. Science 240:185
Davis LG, Dibner MD, Battey JF (eds) (1986) Basic methods in molecular biology. Elsevier Science Publishing Co., lnc., N ew Y ork
Gough JA, Murray NE ( 1983) Sequence diversity among related genes for recognition of
specific targets in DNA molecules. J Mol Biol166:1
Graham A, Steven J, McKechnie D, Harris WJ (1986) Direct DNA sequencing using avian
myeloblastosis virus and Moloney murine leukemia virus reverse transcriptase.
Bethesda Res Lab Focus 8(2):4
205
ever, you should be aware, that diTP destabilizes DNA lowering the
yield. Formamide, added to the polyacrylamide solution, should also
prevent fragments from forming secondary structures during electrophoresis.
• Bands at the same position in 2 or 3 lanes throughout the gel
- Spontaneous deletions may occur during phage growth leading to 2
different template DNAs. Limit the time allowed for growth to 6 hours.
• Faint bands at the bottom of the gel
- If you are primarily interested in the first 50 bp adjacent to the primer,
dilute the dNTP-concentration during the labeling reaction by a third,
increase the ddNTP-concentration by 3-fold, increase the amount of
template and restriet the time allowed for labeling and termination to
1-2 min. Use a short electrophoresis run (1-2 hours).
• Faint bands at the top of the gel
- Increase the concentration of dNTPs by 2-fold and reduce the concentration of ddNTPs by a half during the termination reaction.
• Faint bands all over the gel
- Increase the amount of template and reduce the time for termination
to 1 min. If too long a time is chosen, extraneous nuclease activity may
cause degradation of the newly synthesized DNA strand.
References
Ausubel R, Brent R, Kingston RE, Moore DD, Seidman JG, SmithJA, Struhl K (eds) (1990)
Current protocols in molecular biology, Volumes 1 and 2. Greene Publishing Associates and Wiley-Interscience
Barnes WM, Bevan M, Son PH (1983) Kilo-sequencing: creation of an ordered nest of
asymmetric deletions across a large target sequence carried on phage M13. Methods
Enzymol101:98
Biggin MD, Gibson TJ, Hong GF (1983) Buffer gradient gels and 35 S labelas an aid to
rapid DNA sequence determination. Proc Natl Acad Sei U.S.A. 80:3963
Church GM, Kieffer-Higgins S (1988) Multiplex DNA Sequencing. Science 240:185
Davis LG, Dibner MD, Battey JF (eds) (1986) Basic methods in molecular biology. Elsevier Science Publishing Co., lnc., N ew Y ork
Gough JA, Murray NE ( 1983) Sequence diversity among related genes for recognition of
specific targets in DNA molecules. J Mol Biol166:1
Graham A, Steven J, McKechnie D, Harris WJ (1986) Direct DNA sequencing using avian
myeloblastosis virus and Moloney murine leukemia virus reverse transcriptase.
Bethesda Res Lab Focus 8(2):4