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Familiar with how Sanger sequencing works in theory but want to know how it works in real life? Let me show you how it is done.
Sanger Sequencing has moved from a very manual process using gels to being automated with capillary electrophoresis. As with all technologies, Sanger Sequencing has gotten easier through the years.
So today, let me show you the few steps it takes to set up the Applied Biosystems 3500 Genetic Analyzer. This is a Capillary Array, and it comes with a built-in frame for easy installation. Arrays come in different lengths depending on the desired application -the longer the array typically means you desire a longer read length. Here we have two lengths: 50 cm and 36 cm. 50 cm arrays are usually used in sequencing runs while 36 cm arrays have historically be used for Human Identification applications or fragment analysis. However, now you can use a single 36 cm array for both sequencing and fragment analysis applications on the 3500 series if you’re using the latest data collection software. Today we’re setting up for a standard run, so let’s go with 50 cm.
Next is the polymer pouch and you can choose the correct polymer based off of your desired application. For example, Pop 4 polymer provides fast run and is used more for Human Identification and fragment analysis. POP-6 polymer is ideal for long runs. When used with BigDye v1.1, POP-6 is also ideal for reading bases very close to the 5' end. Pop 7 polymer falls in between Pop 4 and Pop 6, which it is very versatile and designed for a wide range of applications. Today we will be using POP 7 polymer. And this is how you snap that in.
Lastly, these are the Cathode and Anode buffer containers. Unlike the older models of genetic analyzer where you have to dilute the buffers to 1x solutions, the buffers for the 3500 series come pre-packaged and pre-diluted, so all you have to do is remove the seal, add septas to the cathode container and place the buffers onto the instrument. The Cathode buffer goes here and the Anode buffer goes here.
And there you have it, that’s all of the consumables you’ll need for a sequencing run. All these consumables are RFID labelled, so that everything about a run can be tracked from part number, lot number, expiration date, to usage information.
All of the information on the RFID also gets written to the run file so that the output files contain all of that information as well, which makes for less writing in lab notebooks!
So now, all you need to do to get ready for a run is launch the Data collection software. Make sure you have the latest version by the way. Fill the array with polymer, and complete a spatial calibration… and you are ready to go.
I hope that was helpful on setting up a 3500 Genetic Analyzer but I am sure you will have more questions.
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And remember, when in doubt, just Seq It Out
