Guidelines to Sample Preparation and Quantification
As Sanger DNA sequencing was optimum only for pure DNA templates, the purity & amount of DNA template with presence of specific sequencing priming site are crucial to success the DNA sequencing reaction.
You may maximise the success of your DNA Sequencing reaction by having 1st BASE perform the critical Sample Preparation and Quantification steps.
Alternatively, you may learn to quantify and estimate the purity of your DNA template using agarose gel electrophoresis. At 1st BASE, we generate various tools to guide our customers to use agarose gel quantification to make estimation whether the amount of DNA template has enough to success the DNA sequencing reaction. Please contact us for more information.
These are example of agarose gel photos to illustrate the type of DNA templates that require purification after quantification. In some cases, repeat the preparation of the DNA templates (e.g. generating new DNA template) are more efficient than additional purification because all purification steps will reduce the yield of total DNA. Insufficient DNA template are the major cause to fail sequencing reactions.
You may download the guideline of this gel photo directly here.
DNA Sequencing Sample Preparation
We accept PCR products straight off the thermal cycler for DNA purification before sequencing. We accept either individual PCR products in single tubes or 96-well plate format.
Gel Extraction by General Agarose
Instead of purifying the PCR products straight off the PCR reaction, the unpurified PCR products are first run on an agarose gel, then the target DNA band is excised and purified using a silica gel extraction column kit.
We accept transformed bacterial cultures, grow them in our laboratory, and then extract the transformed plasmids using a plasmid prep kit prior to DNA sequencing. Now, we only accept stab culture for individual clones. 96-well format bacterial cultures are accepted in DNA pellet format on dry ice.
Drying of Ready-to-Load Samples
Before adding Ready-to-load samples into load into our Genetic Analyzer, we must add formamide or water into the samples. Residual ethanol on the precipitated BigDye® cycle sequencing will generate relatively low signal data. Hence, additional drying service is required for the customers who are not able to dry their purified BigDye® cycle sequencing reactions completely.
BigDye® cycle sequencing reaction is tolerant to contamination of RNase up to certain level. For DNA template that shows heavy RNA contamination of 1ug or more, it will show relatively low signal data in sequencing. RNase will be used to degrade excess RNA from the DNA template by incubation. Right after RNase treatment, the DNA template will be ethanol precipitated and purified before DNA sequencing.
DNA Sequencing Sample Quantification
By Agarose Gel
This additional quantification service provides customers with a means of verifying and quantifying their DNA template prior to DNA sequencing. A small volume of DNA template is run on an agarose gel to verify its purity and mass before proceeding with DNA Sequencing reactions.
This additional quantification service provides customers with a means of quantifying their DNA template prior to DNA sequencing. A small volume of DNA template is used to measure the concentration and optical density (OD) ratio of 260/280 using the Spectrophotometer.