Frequently Asked Questions about DNA Sequencing
How can I view my sequence data?
To view your sequence data, please download the free ABI Sequence Scanner software.
Other free software is also available for viewing sequencing data;
For Mac users: 4peaks software
For PC users: Chromas Lite (Technelysium)
How much DNA do you need for sequencing?
Standard reactions require a minimum amount of DNA (see below). If you have less than the required minimum, please contact us to discuss your options.
• PCR products (<499 bp): 50-100ng/µL
• PCR products (500-1000 bp): 100-150ng/µL
• PCR products (>1 kb): 60-180ng/µL
• Plasmids: 60-180ng/µL
Please remove primers from PCR products using an Exo1/SAP enzyme clean up method (preferred), column-based purification kit, or gel purification. ACGT can also provide the clean-up at an additional cost per sample. Samples submitted for DNA sequencing should also be free of RNA contamination, salts, EDTA, polyethylene glycol (PEG), and solvents such as ethanol, phenol, and choloroform.
What is your turnaround time for DNA sequencing?
Samples submitted before 3pm can be sequenced by the next business day. Results are typically sent by email before 5pm on the following day.
What is the difference between edited and unedited results?
Unedited results refer to the sequence read that is provided directly by the ABI 3730xl instrument. Edited results are sequence reads that have been reviewed and ambiguities manually edited by a qualified sequencing technician.
My sequencing reads were shorter than I expected, what happened?
Short reads are often an indication that there was too much or too little template DNA in the sequencing reaction. Please be sure to measure the OD 260nm of the samples and submit the proper amount.
Which contaminants in the DNA solution can affect sequencing reactions?
For best results, we request that samples for DNA sequencing be purified (column or gel) and resuspended in water. Contaminants such as RNA, salts, EDTA, polyethylene glycol (PEG), and solvents such as ethanol, phenol, and chloroform, can inhibit the sequencing reaction. The presence of fluorescent molecules can interfere with the emission spectrum of the fluor-labelled terminators.
What is capillary electrophoresis (CE)?
CE is a technique used to separate ionic species based on their size to charge ratio within the confines of a narrow capillary filled with electrolyte.
Which method of DNA sequencing is offered at ACGT?
There are two general methods of fluorescent automated sequencing; dye-labelled primer sequencing (dye is on 5’end of primer oligo) and dye-labelled terminator sequencing (dye attached to terminating dideoxynucleotide triphosphate). ACGT offers the latter method (dye-labelled terminator sequencing) using the ABI 3730xl instrument.
What are BigDyes™?
BigDyes, developed by ABI, are a set of dye terminators labelled with high-sensitivity dyes (1). The dye structures contains a fluorescein donor dye linked to one of four energy-transfer dichlororhodamine (dRhodamine) acceptor dyes. The excitation maximum of each dye label is that of the fluorescein donor and the emission spectrum is that of the dRhodamine acceptor. The BigDye™ terminators are 2-3 times brighter than the rhodamine dye terminators when incorporated in cycle sequencing products.
Do I still get charged for failed reactions?
Failed reactions due to unforeseen problems will be gladly repeated free of charge. Charges will apply to failed reactions due to sample contamination (unpurified PCR products), degraded DNA, or poor primer design.
What LOR (length of read) can I expect?
You can expect to receive at least 600 bases of sequence for successful sequencing reactions.
Primer walking is a technique used to determine the sequence of long DNA fragments (>1 kb). The initial sequence is obtained using a standard primer that hybridizes to the vector sequence or a known sequence within the DNA fragment. Based on the results of the first sequence, a new sequencing primer is designed to prime a second sequencing reaction, thus extending the sequence further downstream. The cycle is repeated until the fragment is sequenced. ACGT does offer a primer walking service.
What is a sample score?
A sample score is generated by the ABI 3730xl sequencer. This number indicates the quality of the read. In order to pass, a sequence must have a sample score greater than 40. This guarantee is only offered as part of the GMP service.
1. Rosenblum BB, et al. New dye-labelled terminators for improved DNA
sequencing patterns. Nucleic Acids Research 1997, 25(22):4500-4504.How can I view my sequence data?