Sequencing Failure

Check template concentration, make sure plates and cartridges are stored at 4° C. 

Take care to avoid crack in gel matrix when loading samples to plate or cartridge.

Dye "blob" or peak at ~ base 70

Unincorporated dye terminators.

The problem of unincorporated dye terminators is very common with use of BigDye® v 3.1.

If using Edge PERFORMA® gel filtration plates or cartridges make sure you centrifuge at the speed specified in the appropriate protocol. Centrifuging too fast can contribute to the appearance of dye peaks or "blobs".

Take care to avoid crack in gel matrix when loading samples to plate or cartridge; applying the sample into the crack may cause the appearance of dye peaks or "blobs".

Use products and protocols developed specifically for BigDye® v 3.1, such as the PERFORMA® DTR V3 96-Well Short Plate, and the PERFORMA® DTR Gel Filtration Cartridges.

You may also want to incorporate the 5X Buffer supplied with BigDye® v 3.1 in the ratios specified by the manufacturer.

If using Edge PERFORMA® DTR  gel filtration plates or cartridges follow speed conditions specified in accompanying protocol.

Suppression of 5' end of sequence

Use Edge PERFORMA® DTR  gel filtration plates and cartridges to purify your samples prior to sequencing.

Noisy background

Use Edge PERFORMA® DTR  plates and cartridges for effective salt removal. If you suspect the template is dirty, re-prepare template.

If you see excessively high G strengths in conjunction with noisy background, repeat sequencing reaction with diluted sample.

Short sequence, loss of 3' end of sequence

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