| Sequencing
Failure
|
Template concentration too
low, use of PERFORMA® DTR gel filtration plate or cartridge that has been frozen, sample applied
to crack in plate or cartridge matrix. |
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. |
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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.
|
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| Suppression
of 5' end of sequence
|
Loss of short sequence fragments.
This is commonly seen when using ethanol purification methods to purify sequencing
reactions. |
Use Edge PERFORMA®
DTR gel filtration plates and cartridges to purify your samples prior
to sequencing. |
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Noisy background
|
Ineffective salt removal, dirty template,
or excessively high signal strength. |
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. |
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Short
sequence, loss of 3' end of sequence
|
Use of ethanol precipitation
or other inferior method to clean up sequencing reaction, especially when using
very low dye volumes (eg.:0.33 µl of Big Dye® v 3.1 in a 5 µl
sequencing reaction). |
Use Edge PERFORMA®
DTR plates and cartridges to purify your sequencing reaction. |
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