226
- Amplicons that are less than 500 bp can be run with a shorter
extension time (for example 2 min). - dNTPs, salts, and unincorporated dye terminators can obscure
data at the beginning portion of the sequence which can ulti-
mately interfere with base calling. - For effective cleanup, it is essential to keep all materials
homogenous. - Ensure that you do not skip the drying step. Samples that are
not fully dried will affect sequencing results. - Do not heat samples!
- Run the sample immediately.
- Sequence signal intensity should range between 700 and
6000. Signal below 700 may be affected by background and
adjacent sequences while signal of over 6000 may produce
overload/poor reads. - During examination of the electrophoretogram, aware that
heterozygotic mutations may not be properly labeled by an
ambiguity code if the signal strength from the alternative allele
is too weak. A manual inspection of the electrophoretogram
for any dual peaks or other anomalies following BLAST is
highly recommended.
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