Reactions - Cycle Sequencing


1. 4.4 μl ddH2O
2. 1.3 μl “5x replacement buffer”
3. 2.0 μl 2μM primer
4. 0.3 μl sequencing mix (ABI BigDye v3.1)

Mix and add 8 μl of master mix to each reaction. Add 2 μl purified DNA (approximately 20-50 ng/μl for standard 500-2000 kb pieces).


1. 96ºC for 60 sec. (heat)
2. 96ºC for 10 sec. (denature)
3. 50ºC for 15 sec. (anneal) – primer specific temp
4. 60ºC for 75 sec. (extend)
5. repeat steps 2-4 15 times
6. 96ºC for 10 sec. (denature)
7. 50ºC for 15 sec. (anneal) – primer specific temp
8. 60ºC for 90 sec. (extend)
9. Repeat steps 6-8 5 times
10. 96ºC for 10 sec. (denature)
11. 50ºC for 15 sec. (anneal) – primer specific temp
12. 60ºC for 120 sec. (extend)
13. Repeat steps 10-12 5 times
14. 10ºC for ever (hold)

You may want to increase the total number of cycles to 35 for sequencing reactions with low DNA concentrations (currently there are 25 cycles). You may also want to use 42ºC annealing temperature; it works consistently on all primers, although many primers can be done at higher temperatures. Basically lower annealing temperatures do not matter as long as you do not have alternate annealing sites. This protocol is based on Platt et al. (2007).


Cycle sequencing 5x replacement buffer (1 liter)


400 ml 1M Tris-HCl pH 9.0
10 ml 1M MgCl2
590 ml ddH20

Stir for minimum of 1 hour. Take and record conductivity. Filter into 1 L storage contained. Aliquot into 15 ml Falcon tubes as necessary. Store at room temperature or 4°C.

 

Reference:

Platt, A. R., Woodhall, R. W. & George Jr., A. L. (2007). Improved DNA sequencing quality and efficiency using an optimized fast cycle sequencing protocol. Biotechniques 43, 58-62. doi:10.2144/000112499

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