Great turn around time on my 80 exome project!
Yes. A few hours on the bench or overnight storage will not damage performance or reduce shelf life as long as the cartridges are snapped shut and the zip seal foil bag is properly closed. We do not recommend long-term storage at room temperature.
Unincorporated dye terminators comigrate with sequencing products during electrophoresis. The most common peaks occur roughly 70 bases into the sequencing run, and in cases of severe contamination, additional peaks may appear in the vicinity of base 120, 220, or higher. If you are experiencing difficulties with unincorporated dye terminators after using our purification products, please refer to our Trouble Shooting Guide for suggestions on how to eliminate these problems.
The centrifugation time recommendations are based on time the centrifuge starts spinning. As most centrifuges that accommodate micro centrifuge tubes accelerate to full speed within seconds, such delays are likely to have minimal impact on performance. If your centrifuge takes more than 15 seconds to reach full speed, it may be necessary to adjust your spin conditions. However, we have not observed that slow acceleration in a microcentrifuge significantly alters column performance.
a. Yes, but we recommend that you dilute the BigDye® to 1 µl or less to insure optimum performance. Be sure to use sequencing reaction diluent, rather than water, to reduce the concentration of BigDye® version 3.1 in your sequencing reaction.
b. If you are using BigDye® v. 3.1, and experiencing dye blobs, pre-spin the cartridges at 850 x g for 3 minutes, transfer the cartridge to a clean tube, load the sample onto the cartridge and spin at 850 x g for 3 minutes.
Pre-spin the cartridge at 750 x g for 2 minutes, transfer the cartridge to a clean tube, load the sample onto the cartridge and spin at 750 x g for 2 minutes. If you are using BigDye® v. 3.1, and experiencing dye blobs, pre-spin the cartridges at 850 x g for 3 minutes, transfer the cartridge to a clean tube, load the sample onto the cartridge and spin at 850 x g for 3 minutes
Spin the cartridge at the correct speed for an additional minute. You might need to dry the sample and resuspend it in 10-20 µL water. Even though this procedure might "save" your sample, it will not be optimal and you may need to re-run the sequencing reaction.