Dye peaks present

Complete removal of ethanol is critical for successful cleanup. Residual ethanol can be removed by aspirating at a pipet setting of 150 µl or higher, coupled with slow aspiration. There should be no need for a second aspiration. However, if a second aspiration is necessary, care should be taken to ensure that ethanol is not reintroduced into the wells from the pipet tip.

Increase the number of washes from 2 to 3 washes to ensure that all salt and dye have been removed.

Increase the number of washes from 2 to 3 washes to ensure that all salt and dye have been removed.

More than 4 µl BigDye® used per sequencing reaction.

Use lower dye concentration. Dilute the dye with the appropriate sequencing buffer.

Increase the number of cycles during thermal cycling.

Increase the concentration of the template in the sequencing reaction.

Optimize template and primer concentrations. Consult dye manufacturer's recommendations.

Start with high purity template.

Check thermal cycler for program entry error.

Check for potential machine failure.

Check ethanol concentration used. Be certain that freshly prepared 80% ethanol is used for the washes.

Use at least 100 µl of freshly prepared 80% ethanol per wash.

Use Edge BioSystems' MagWell Magnetic Separator.

Optimize the protocol for use with an alternative magnet.

Avoid prolonged exposure to light.

Avoid extended storage at temperatures above 4°C.

Store post purified samples at -20°C.

Low recovery (Low signal)

Aspirate all ethanol. Reduce the drying time at room temperature to 3 minutes. Resuspend immediately.

Aspirate all ethanol. Heat at 98°C for30 seconds. Resuspend immediately.

At the elution step, add recommended volume of deionized water. Incubate for one minute at room temperature, then pipet mix to resuspend

Use recommended volume of deionized water. Incubate for one minute at room temperature, then pipet mix to resuspend.

Vortex sample at medium speed for at least30 seconds to properly resuspend the samples.

In the absence of a vortex, pipet aggressively.

Direct elution with formamide is not recommended.

Use recommended volume of deionized water. Incubate for one minute at room temperature then pipet mix to resuspend.

If the sample must be in formamide, the sample can be eluted in water then dried down and resuspended in formamide.

Check ethanol concentration used. Be certain that 100% ethanol is used for precipitation

Be certain that the correct volume of 100% ethanol was used.

Check ethanol concentration used. Be certain that freshly prepared 80% ethanol is used for the washes.

Try to remain within the suggested number of washes unless your reaction was done with greater than 4 µl BigDye®, then up to 3 washes may be required.

Check ethanol concentration used. Be certain that freshly prepared 80% ethanol is used for the washes.

Increase the number of washes from 2 to 3 washes to ensure that all salts and dye have been removed.

Start with high purity template.

For optimal performance, use Edge BioSystems' Plasmid 96 MiniPrep Kit for plasmid purification and Edge BioSystems' Quickstep™2 DNA Extraction Kit for PCR purifications.

Use Edge BioSystems' MagWell Magnetic Separator.

Optimize the protocol for use with an alternative magnet.

Noisy background on the sequence

Check ethanol concentration used. Be certain that freshly prepared 80% ethanol is used for the washes.

Increase the number of washes from 2 to 3 washes to ensure that all salt and dye have been removed.

Follow the steps recommended for troubleshooting low signal sequences.

If the signal is high, dilute the sample before loading on the sequencer.

Check thermal cycler program for entry error.

Check thermal cycler for mechanical or electrical failure

Troubleshoot your thermal cycler to ensure optimal conditions for preparing reactions.

Shake well or vortex the resin prior to use.

If using a robotic workstation, make sure to pipet mix the resin prior to each aspiration step.

If using reagent reservoir, rock back and forth 8-10 times to resuspend resin before pipetting.