(last update : 15-05-2009)
Sequencing protocol for BigDye 3.1 on ABI 3130XL genetic analyzer
- Cleaning of PCR product
You need 1,25 µl cleaned PCR product for each sequencing reaction. If you have to sequence several reactions of one PCR product, adjust the volumes.
For each reaction : pipet into a reaction tube :
- PCR product : 3 µl
- exonuclease I (10 U/µl) : 0,6 µl
- shrimp alkaline phosphatase (2 U/µl) : 0,6 µl
Mix, spin down and incubate 15 minutes at 37°C, followed by 15 minutes at 80°C to inactivate the enzymes.
- Cycle sequencing
Bring into a reaction tube the following products :
- Terminator Ready Reaction Mix : 4 µl (can be diluted : see bottom of this page)
- cleaned PCR product : 1,25 - 5,5 µl (10-40 ng PCR product or 150-300 ng double-stranded DNA)
- primer (5µM) : 0,5 µl
- sterile water : add until total volume is 10 µl
cycle sequencing :
29 cycles : 30 seconds 96°C - 15 seconds 50°C - 2 minutes 60°C
- Precipitation
- add to every tube 26 µl (95 % ethanol + 0,12 M NaAc), mix and put at roomtemperature for 15 minutes
- centrifuge 15 minutes at 14.000 rpm (4 °C)
- remove supernatans (pellet is invisible)
- add 125 µl 70% ethanol to each tube, mix gently
- centrifuge 5 minutes at 14.000 rpm (4 °C)
- remove supernatans (pellet is invisible)
- dry 15 minutes under vacuum
(These samples can be stored at -20 °C)
- Loading of the samples
- add 10 µl formamide to each tube
- transfer to a 96-well plate
- spin down
- heat the samples for 2 minutes at 95°C and put immediately on ice
- run on the sequencer
Dilution of the BigDye mix
8 times diluted reaction mix :
- sterile water : 35 µl
- 5x BigDye dilution buffer 35 µl
- BigDye mix : 10 µl
Use 4 µl of this for each sequencing reaction.
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