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The two techniques involved different starting DNA amounts, PCR conditions, agarose gel concentrations, sample placement on the gels, lane size, number of lanes per gel, and duration of electrophoresis.
We used GeneScan analysis software, an internal lane size standard, and an ABI PRISM 377 DNA sequencer.
Genescan 600-Liz (PE Applied Biosystems) was used as an internal lane size standard.
An internal lane size standard was added to the PCR samples for accurate sizing of alleles and to adjust for run-to run variations.
Sixteen primer pairs correctly amplified with the expected size and were run on an ABI 3730 automated sequencer (Applied Biosystems, Foster City, California, USA) using LIZ 500 as the internal lane size standard.
Allele sizing was carried out by automated fluorescent scanning detection in an ABI 377XL DNA sequencer (Applied Biosystems) using ROX500 as internal lane size standard, and the software GENESCAN and GENOTYPER Applied Biosystemss).
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Samples were prepared for genotyping by combining 0.7 to 1.0 uL of the diluted amplicons with 8 uL diluted GeneScan ROX500, the internal-lane size standard.
Each lane included an internal-lane size standard labelled with ROX dye (Applied Biosystems) and GeneScan 3.1 fragment analysis software (Applied Biosystems) was used for fragment size determination and pattern analysis.
10 μg of crude cell lysate (1, 3) and eluate obtained after IMAC purification (2, 4) were loaded in each lane; protein size markers (M).
b PCR with FADO primers on the cDNA of transformants 1 7 (lanes 1 5, 7 and 8) and with the FAR primers (lanes 9 15); 100 bp DNA ladder on lane 6 (size in base pairs).
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