Data on single-step purification method for dye-labeled DNA sequencing

Dye-labelled DNA sequencing is one of the most common and robust technique required for molecular biology since 1977 (Sanger, 1977) [1]. I have recently provided the single-step purification method for dye-labeled sequencing products, which is based on the removal of the washing step in EDTA/ethanol precipitation (Fujikura, 2015) [2]. Here I assess and report the accumulated data of the modified method on the larger scale in practice.


a b s t r a c t
Dye-labelled DNA sequencing is one of the most common and robust technique required for molecular biology since 1977 (Sanger, 1977) [1]. I have recently provided the single-step purification method for dye-labeled sequencing products, which is based on the removal of the washing step in EDTA/ethanol precipitation (Fujikura, 2015) [2]. Here I assess and report the accumulated data of the modified method on the larger scale in practice. &

Experimental features
The modified EDTA/ethanol method skips the washing step in EDTA/ethanol precipitation for dye-labeled DNA sequencing.

Data source location
Japan

Data accessibility
The data are with this article.

Value of the data
The data provide the scheme and characterize the success rate for modified EDTA/ethanol purification method for dye-labeled DNA sequencing.
The data provide the optimization of methods, including the amount of DNA template, primers, and buffer conditions for rapid purification of dye-labeled sequencing.
The modified method provides the quick and inexpensive purification technique for DNA sequencing.

Sequencing reaction
Sequencing reaction was performed in a 10 μl scale using the BigDye Terminator v3.1 Cycle Sequencing Kit (dilution rate: 1:16-1:32) (Applied Biosystems, Foster City, CA, USA), 10 pmol of various primers, and 50-1500 ng of template DNA (50 ng for 100-300 bp PCR product, 250 ng for 300-2000 bp PCR product, or 1500 ng for plasmid) for one experimental run. The following thermal cycle was used for the amplifications: 96°C for 1 min, followed by 40-50 cycles of 96°C for 10 s, 50-58°C for 5 s and 60°C for 150 s.

Modified purification technique and data analysis
1 μl of 125 mM of EDTA and 30 μl of 100% ethanol were added to each sequencing reaction, and the mixture was then lightly vortexed (Fig. 1). After centrifugation (15,000g, 5 min for tube; 4000 g, 15 min for plate), the supernatant was carefully aspirated and discarded. Next, after drying the precipitate, 40 μl of distilled water or TE0.1 buffer (10 mM Tris-acetate [pH 8.0] þ 0.1 mM EDTA) was added to each reaction. ABI PRISM 3130 Genetic analyzer and POP-7 polymer were used as the separation machine and matrices, but the other Genetic analyzer (ABI Prism 310, 3730, or 3500) and polymers (POP-4 or POP-6) are also applicable. The sequence data was analyzed with ABI Prism DNA Sequencing Analysis Software v5.1. The quality value score 20 (QV20) was used as indicator of sequencing quality. Student's t tests were performed to evaluate the significance between two methods.