Dilute primers to 100 uM by adding 10x # of nmol H2O
Working stock (5 uM): 2.5uL stock, 47.5uL dH2O
PCR
MM1
|
Comp
|
1x Volume
|
40x
|
Final Conc
|
|
Water
|
6 μl
|
240
|
|
|
dNTPs
|
.5 μl
|
20
|
200 μM
|
|
R primer
|
2.5 μl
|
100
|
0.5 μM
|
|
F primer
|
2.5 μl
|
100
|
0.5 μl
|
MM2
|
Water
|
9.17 μl
|
366.8
|
|
|
10x buffer
|
3.2 μl
|
128
|
2 mL MgCl2
|
|
Taq
|
.13 μl
|
5.2
|
1.25 U/rxn
|
Add 1 μl DNA to each tube
|
Step
|
Cycles
|
Time
|
Temp
|
|
Initial Denaturation
|
1
|
2 min
|
94 deg
|
|
Denaturation
Annealing
Elongation
|
1-8
|
30 s
1 min
1 min
|
94
54,53,52,51,50,49,48,48
72
|
|
Denaturation
Annealing
Elongation
|
9-33
|
30 s
1
1
|
94
48
72
|
|
Final Elongation
|
1
|
7
|
72
|
|
forever
|
10
|
SAP/Exo
MM
|
Reagent
|
1x
|
13x
|
| Sap |
0.5 ul
|
6.5
|
|
Exo
|
0.5 ul
|
6.5
|
5 ul of PCR product + 1uL MM
|
Step
|
Time
|
Temp
|
|
Incubate
|
30 min
|
37 C
|
|
Denature
|
15 min
|
80 C
|
|
Chill
|
forever
|
25 C
|
Sequencing
Getting My Genes Wet 
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