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Thanks to the new BigDye Direct Cycle Sequencing Kit it's easy to imagine a simpler sequencing process. Because now, fewer steps and a faster workflow actually yield superior results.
Sanger sequencing is the gold standard against which all other sequencing technologies are compared. It's good.
Here's a way to make the process even better. With BigDye Direct you can speed and simplify your workflow, and read as close as one base from the primer. You have other things you need to do, and the fewer steps the better.
We start with amplifying the template DNA with a PCR step. We will use these same two tubes all through this process.
Prepare the PCR using the BigDye Direct PCR Master Mix. The reaction volume is 10 microliters.
Place the tubes in a thermal cycler. We recommend the Veriti Thermal Cycler, and perform the PCR in only 64 minutes.
Now it's time for the PCR clean-up and cycle sequencing in the same step, add 2 microliters of the BigDye Direct Sequencing Master Mix to both reaction tubes. Then add 1 microliter of the BigDye Direct M13 forward sequencing primer to one reaction tube, and 1 microliter of the BigDye Direct M13 reverse sequencing primer to the other reaction tube.
Perform the sequencing reaction, this time placing the tubes in the Veriti Thermal Cycler for only 80 minutes.
Now, at this point you may be asking yourself "Wait—didn't they skip a step? What about the PCR clean-up that's normally performed before the sequencing reaction?"
Well, BigDye Direct is unique in that the sequencing master mix contains the PCR clean-up reagents AND the sequencing reagents. During the sequencing reaction, the PCR clean-up reagents digest unincorporated PCR primers but do not affect the protected BigDye Direct M13 sequencing primers.
So your sequencing reaction can now be performed in optimal conditions.
Getting back to the process, once the PCR clean-up/sequencing step is complete it's time to purify the sequencing reactions prior to electrophoresis. Just add the reagents from the BigDye Xterminator purification kit into the reaction tubes and vortex for 20 minutes.
The samples are then ready to be analyzed using your Genetic Analyzer System. This is where BigDye Direct offers another significant advantage. When using the POP-7 polymer, the time to read the electropherogram takes place in about half the time, and it also results in higher-quality data.
Using the standard process, the first bases of the sequencing are unreadable.
They can be read when using BigDye Terminator 1.1 and a POP-6 polymer, but doing so doubles the migration time.
However, with BigDye Direct and POP-7, you can easily read the first base after the gene specific primer sequence, allowing you to quickly analyze your sample and achieve better-quality data.
Let's take a quick look again at the complete BigDye Direct process.
PCR and PCR clean-up, along with sequencing reaction and purification, are all performed in the same reaction tubes, meaning there's less risk of error, and you can easily track your tubes from beginning to end.
All in all giving you:
• Fewer steps,
• A faster workflow, and
• Superior results.
Imagine a simpler sequencing process.
Imagine the BigDye Direct Cycle Sequencing Kit.