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Protocol for DNA sequencing in PCR tubes

(modified from the PE ABI BigDye Ready Reaction Termination Mix protocol)

1) Dilute the 10µM stock solution of sequencing primer 1:15 with sterile dH2O. Pipette 2µl of the diluted sequencing primer into appropriately labelled, thin-walled PCR tubes.

2) Pipette 2µl of the BigDye Ready Reaction Termination Mix into each tube.

3) Add 1µl of each PCR product to the appropriate tube, ensuring that all 3 reaction components are mixed together. These are 1/4 size reactions (5µl total reaction volume compared with 20µl total reaction volume suggested in the ABI BigDye sequencing manual).

4) Run the following cycling program:

Step Temperature /°C Time
1 96 10 s
2 50 5 s
3 60 2 min
4 repeat steps 1, 2 and 3 a further 29 times
5 4 forever

5) Remove the tubes from the thermal cycler. Add 15µl of sterile dH2O to each tube to make up the reaction volume to 20µl (this helps to prevent severe dye blobs).

6) Add 2µl of 3M NaOAc, pH 4.6, and 50µl EtOH to labelled 1.5ml eppendorf tubes. Add each 20µl sequencing reaction to the appropriate tube containing the salt/EtOH mixture. Vortex briefly to mix. Incubate at room temperature for 15 min.

7) Spin the tubes for 15-30 min at maximum speed. Immediately after spinning, remove the supernatant using a pipette.

8) Wash the pellets by adding 500µl of 70% EtOH to each tube and spin again at maximum speed for a further 10 min. Carefully remove as much of the EtOH as possible (this reduces dye blobs in the sequencing reactions) and dry the pellets in the vacuum dryer (medium rate). The dried pellets can be stored at -20oC until ready for loading on a sequencing gel (resuspend each pellet in 3-4µl of loading buffer on the same day as they are to be run).

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This publication made use of the Neisseria Multi Locus Sequence Typing website (https://pubmlst.org/ neisseria/) sited at the University of Oxford (Jolley et al. Wellcome Open Res 2018, 3:124 [version 1; referees: 2 approved]). The development of this site has been funded by the Wellcome Trust and European Union.