|
Cycle Sequencing using the 'Promega fmol®' Kit
[Promega fmol® DNA Cycle Sequencing System, Promega UK Ltd, Delta
House, Chilworth Research Centre, Southampton SO16 7NS, UK]
1. a) Prepare a set of four tubes per isolate to
be sequenced, and label tubes T, C, G and A.
A thin-walled 96 well microtitre plate may
also be used.
b) Add 1 µl of the appropriate dd/dNTPs into
each tube/well (in the order T, C, G, A)
c) Spin briefly in the microfuge and store at 4°C
until needed (see Electrophoresis of Cycle
Sequencing PCR Products).
2. For each set of reactions add in a separate tube the following:
Reagent
Amount (µl)
|
Template DNA
|
|
|
|
|
(Wizard Prep™ purified)*
|
3
|
5
|
10
|
|
5 x sequencing buffer
|
4
|
4
|
4
|
|
32P-ATP labelled primer
|
1.5
|
1.5
|
1.5
|
|
Sequencing grade Taq
|
|
|
|
polymerase (5 U/µl)
|
1
|
1
|
1
|
|
DEPC water
|
10.5
|
8.5
|
3.5
|
|
Total
|
20
|
20
|
20
|
* the amount of template used will depend on band intensity/concentration.
Vary volume of water accordingly.
3. Mix and spin
4. a) Add 4 µl of mixture to each dd/dNTP
tube/well previously prepared.
b) Spin down (use a flat bed centrifuge if using a
96 well plate, at 1000 rpm maximum)
5. Add a drop of mineral oil on top of reaction mixture.
6. Put onto a pre-heated thermocycler at 94°C.
Running conditions:
|
94°C
|
2 min
|
1 cycle
|
|
92°C
|
1 min
|
30 cycles
|
|
55°C
|
1 min
|
|
|
72°C
|
90 s
|
|
|
20°C
|
Hold
|
1 cycle
|
7. Add 4 µl of stop solution (see table below) to each tube/well,
and spin down.
8. Heat at 80-90°C for 2-5 minutes before running on a gel.
9. Load 2.5-3.5 µl into each well.
Fmol® Sequencing Stop Solution
|
Reagent
|
Final conc.
|
Amount (µl)
|
|
Formamide
|
95%
|
950
|
|
2M NaOH
|
10 mM
|
5
|
|
10% Bromophenol Blue
|
0.05%
|
5
|
|
10% Xylene Cyanol FF
|
0.05%
|
5
|
|
DEPC water
|
|
35
|
|
Total
|
|
1000
|
|
