Cycle Sequencing


We use the Prism Cycle-Sequencing kit from Perkin-Elmer/ABI (DNA Sequencing Kit, Dye Terminator Cycle Sequencing Ready Reaction, Part Number 402122).  This kit is for dye terminators, not dye primers.  Generally, when we receive a new box of Prism, we break up the tube into 3-5 additional tubes in order to reduce the chance of contamination.  An X is marked on the active tube, please use this one first.

Cycle sequencing in theory works the same way as the Sanger di-deoxy reaction whereby the nucleotide terminator is labeled with 1 of 4 florescent dyes corresponding to A, T, C, or G.  Since you can only sequence one strand of the double-stranded PCR product per reaction, you will only add ONE of your diluted PCR primers.

The reaction volumes below are some variation of what people in the lab use.  Talk to someone in the lab about what best suits your purposes:
Volume
What
 
   
3 - 6 ul
DNA template
from PEG-3350 cleanup procedure
3 - 5 ul 
Sequencing Primer
 (2.5 uM or 2.5 pM/ul) 
basically a 4-fold dilution of the PCR primer (10uM)
4.5 - 6 ul 
Prism
Perkin-Elmer commercial kit
3 -5 ul 
10 mM Tris buffer 
*optional *
 
13-18 ul total



Perkin-Elmer recommends 8 to 9ul of Prism, but this amount is not necessary for our purposes.  Template concentration will have to be ascertained in some consistent method such as agarose gel quantification following PEG purification or experience with the method.

The standard, cycle sequencing conditions are loaded on each of the Perkin-Elmer thermal cyclers (2400, 9600, & 9700).
 
Temp (°C)
Time
Cycles
96°C 
1:00 
1 cycle
96°C 
50°C 
60°C 
0:10 
0:05 
4:00
25 cycles
4°C  
Soak
There is no reason to alter these conditions. 



Now proceed to Sephadex G-50 clean-up of sequencing reactions