Now that your PCR product has been cloned, you are ready to carry out cycle-sequencing.
|– PCR tubes
– ddH2O 5.2 Î¼l(amount will vary, depending on amount of template)
– Template 2.0 Î¼l (~7ng of cleaned PCR or colony PCR product; Î¼l
– Primer (10Î¼M) 0.30 Î¼l depends on concentration)
– Buffer 1.5 Î¼l
– Big dye 1.0 Î¼l
Total volume = 10 Î¼l
1. Set up the BigDye Reaction in a small 0.5 ml microcentrifuge tube.
A. There will be one tube for each direction (forward and reverse).
B. Label each tube with the sample name and direction, e.g. sample1F and sample1R.
2. Add 5.2 Î¼l sterile water to the PCR tube. Touch the tip to the side or bottom of the PCR tube to make sure it all goes into the tube.
3. Add 1.5 Î¼l of 5X big dye buffer (clear solution) to the water in the PCR tube. When adding the buffer, touch the tip of the pipettor to the top of the water already in the tube.
4. Add .3 Î¼l of one primer. Be sure to touch the tip to the liquid already in the PCR tube before pushing the primer out of the tip.
5. Add 2 Î¼l of your PCR product. Again, put the tip of the pipettor into the liquid already in the PCR tube before pushing the liquid out of the pipettor.
6. Add 1 Î¼l of BigDye to the liquid in the PCR tube.
7. Place your tube in the thermal cycler and use this thermal profile:
- 96C 10 sec – denature DNA
- 50C 5 sec – allow primer to anneal (stick)
- 72C 4 min – strand extension
- Repeat a-c for 25 cycles.
- 4C forever (to refrigerate samples)
8. Clean up samples with preferred method and submit for sequencing.