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