Esophageal Adenocarcinoma Methods and Protocols

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  1. Amplicons that are less than 500 bp can be run with a shorter
    extension time (for example 2 min).

  2. dNTPs, salts, and unincorporated dye terminators can obscure
    data at the beginning portion of the sequence which can ulti-
    mately interfere with base calling.

  3. For effective cleanup, it is essential to keep all materials
    homogenous.

  4. Ensure that you do not skip the drying step. Samples that are
    not fully dried will affect sequencing results.

  5. Do not heat samples!

  6. Run the sample immediately.

  7. 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.

  8. 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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Katherine T. W. Lee et al.
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