Setting up Sequencing Run S0592

Today at 454 Bio Jinchun set up our 592nd sequencing run

Today at 454 Bio Jinchun set up our 592nd sequencing run: S0592.

This was a standard sequencing run- Jinchun used all of the protocols laid out in Docs to prepare the sequencing reservoir for sequencing on the Mk3 Transformer. To record experimental details about this run, Jinchun filled in the helpful Seq-Setup-Worksheet:

All of the materials and reagents were prepared ahead of time. Solutions were made for hybridizing the sequencing primer, loading Therminator polymerase, and sequencing. Jinchun pre-heated the Transformer to 65 C.

Once the transformer reached 65 C, Jinchun hybridized the sequencing primer. This process takes about 20 minutes: 10 minutes on the pre-heated Transformer, then 10 minutes on the benchtop. Once the sequencing reservoir goes from Transformer to benchtop, Jinchun adjusted the temperature on the Transformer to 58 C for the sequencing run.

After hybridizing the sequencing primer, Therminator was pre-loaded onto sequencing clusters. This quick incubation step was followed by a rinse with OPB (One Pot Buffer). This rinse is especially critical if DISCS system is being used. Any unbound Therminator remaining in solution has the potential to extend DISCS templates with Lightning Terminators™; rinsing minimizes the chances of this happening.

Once Therminator was loaded and rinsed, Jinchun carefully added 400 µL of Sequencing solution to the reservoir to ensure no bubbles formed. The reservoir was quickly inserted into the Transformer and locked into place. Lightning Terminators™ immediately begin incorporating, so Jinchun was able to set the focus positions for each LED during the 10 minute cycle 1 incorporation incubation.

When the 10 minute incubation was just about finished, Jinchun double checked all of the focus positions still showed fluorescent sequencing clusters sharply in focus. As soon as his timer went off he hit “Start Protocol” and the Transformer took over the sequencing run. Jinchun went and got some lunch at Pa’s Place.