Preparation of 100 Base pair DNA Standard
Reaction
The set-up for multiple tubes:
Reagent
H2O
Taq Buffer, 10X
Mg2+, 25 mM
dNTP mix, 10mM
Forward Primer
Reverse Primer
Taq Polymerase, 5U/µL
pBluescript
Totals
For 100 µL
72
10
10
4
1.5
1.5
0.5
0.5
100
x 8 Tubes
576
80
80
32
(12)
(12)
4
4
800
x 2 Reactions
1152
160
160
64
(24)
(24)
8
8
1600
Of which, 97 µL (reagents less primers) goes into each tube, followed by 1.5 µL each of the appropriate
primers.
Primers
Forward
ROXF
ROX401F
Reverse
ROX104
ROX200
ROX305
ROX508
ROX597
ROX738
ROX401
ROX305
Resulting Fragment (bp)
104
200
305
508
597
738
401
800+
Only two forward primers are used. The last set is an optional addition to give an ~800bp fragment.
Cycling Protocol
T (°C)
94
94
Melting
Time (min.)
4
1
Hold
Anealing
T (°C) Time (min.)
50
2
56
2
4 °C
Extension
T (°C) Time (min.)
72
2
72
2
Forever
Repeats
33
Cleanup & Assembly
Check each reaction on a gel for fragment intensity. Re-run any failed reactions.
Combine all reactions in a 2 ml centrifuge tube (total is ~ 600 µL).
Add 60 µL of 3M sodium acetate and 1200 µL of 100% ethanol.
Incubate overnight at -20 °C. (optional)
Spin for 20 minutes at 10,000 rpm. Discard supernatant. Wash pellet with 1 ml 70% ethanol.
Spin for 20 minutes at 10,000 rpm. Discard supernatant. Let pellet air dry.
Resuspend in 500 µL of TE buffer (sterile).
Aliquot into 50 µL samples and store at -20 °C. Use ~ 2 µL/gel lane.