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Protocol for DNA sequencing in microtitre plates

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

1) Orient each microtitre plate used by marking the top left corner with permanent ink.

2) Dilute the 10µM stock solution of sequencing primer 1:15 with sterile dH2O. Using an automatic multi-dispensing (multi-channel, if appropriate) pipette, dispense 2µl of the diluted sequencing primer into the bottom of the wells of the microtitre plate.

3) Use the automatic pipette to dispense 2µl of the Big Dye Ready Reaction Termination Mix into all of the used wells of the microtitre plate.

4) Add 1µl of each PCR product to the appropriate well of the plate, 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).

5) Seal the microtitre plate using an adhesive cover slip. Place in the thermal cycler and close the lid, ensuring that the heated plate inside the lid is in contact with the top of the plate, by rotating the screw on the top of the lid until tight.

6) Use the following thermal cycler 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 24 times
5 4 forever

7) Remove plate from thermal cycler and place in a 'plate holder', before carefully peeling back the adhesive cover. Use the multi-channel, multi-dispensing pipette to add 15µl of sterile dH2O to each well of the microtitre plate to make up the reaction volume to 20µl (this helps to prevent severe dye blobs).

8) Mix 7.0ml EtOH with 280µl of 3M NaOAc, pH 4.6, in a small reagent reservoir. Use the multi-channel, multi-dispensing pipette to add 52µl of the EtOH/salt mix to each of the wells of the microtitre plate. Re-seal the plate and vortex briefly to mix. Incubate at room temperature for 45 min.

9) Spin the microtitre plates for 1 hour at 2,750xg (4°C). Immediately after spinning, remove the adhesive cover and invert the plate on white tissues. If this is not done immediately, spin the plates for another 2 min before discarding the supernatant. Place the inverted plate onto a piece of Whatman 3MM chromatography paper and spin again at 500xg for 1 min, to remove any residual EtOH from the wells.

10) Use the multi-channel pipette to add 150µl of 70% EtOH to each of the wells of the microtitre plate. Re-seal the plate and spin again at 2,750xg for 10 min. Immediately after spinning, remove the adhesive cover and invert the plate on white tissues. Place the inverted plate onto a piece of Whatman 3MM chromatography paper and spin again at 500xg for 1 min. The pellets are now dry and the plates can be sealed and stored at -200°C 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 Campylobacter Multi Locus Sequence Typing website (https://pubmlst.org/ campylobacter/) 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.