DyeTerminator removal for Capillary Sequencer (ABI3100) - Edg e

PBO (Peter Bjarke Olsen) pbo at novozymes.com
Thu Jul 5 08:48:34 EST 2001


Hi Jarmo,

We use the Milliporesystem with good results. We do the following.

1:Add 300 ul water
2: Let the sephadex swell for 60 minutes.
3: Centrifuge 3 min at 1900Xg
4: Add 200 ul water
5: Centrifuge 3 min at 1900xg
6: Add sample and centrifuge 3 min at 1900xg .
7: The samples are loaded directly.

We dilute the bigdye terminitors (v2) 4 times and we do 50 pcr cycles.
In general we see only small amounts of dye blobs. As other have metioned it
is important to load the sample in the middle of the wells.
>From may to june 2001 we produced 14232 sequences (54 different users or
projects).

Average phred20: 490
Highest phred20 score: 788
Lowest phred20 score: 0
Average signal: 267
Highest signal: 3023
lowest signal 2.75

We use a 3700 so I don't know if the signals can be compared.
Has anyone tried to use this system (or a similar gelfiltration system) with
version 3 of the Bigdyes? Applied Biosystems have told me that it doesn't
work.

Best regards,

Peter Bjarke Olsen





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