From Powers Lab UC Davis
|
Cocktail |
n* ul | H20 (variable depending on amt. of MnCl2) |
| 2.0 ul | 10x Taq Buffer | |
| 1.0 ul | Primer #1 (10 uM) | |
| 1.0 ul | Primer #2 (10 uM) | |
| 1.0 ul | Plasmid DNA (template) | |
| 6.0 ul | 50 mM MgCl2 | |
| 0.5 ul | Taq enzyme | |
| 2.0 ul | 2mM dNTP's ( [dATP] is variable, see table) | |
| x* ul | 5mM MnCl2 (variable, see table) | |
| 20 ul | Total volume |
| PCR Conditions |
Step
|
Temp
|
Time
|
| (1-2kb fragment) |
1)
|
95 C
|
3'
|
|
2)
|
95 C
|
45 sec
|
|
|
3)
|
50 C
|
30 sec
|
|
|
4)
|
72 C
|
2'
|
|
|
5)
|
Repeat 2)
x 25
|
||
|
6)
|
72 C
|
10'
|
|
|
7)
|
4 C
|
Forever
|
|
|
PCR
Mutagenesis Results
|
||||
|
Group
|
Final [MnCl2]
|
Final [dATP]
|
Mutations
(out of 600bp)
|
Percentage
|
|
I
|
0mM
|
.2mM
|
0
|
0.00
|
|
I
|
0mM
|
.2mM
|
0
|
0.00
|
|
II
|
0.2mM
|
.1mM
|
3
|
0.50
|
|
II
|
0.2mM
|
.1mM
|
2
|
0.33
|
|
III
|
0.2mM
|
.2mM
|
2
|
0.33
|
|
III
|
0.2mM
|
.2mM
|
4
|
0.67
|
|
IV
|
0.4mM
|
.1mM
|
10
|
1.67
|
|
IV
|
0.4mM
|
.1mM
|
12
|
2.00
|
|
V
|
0.4mM
|
.2mM
|
7
|
1.17
|
|
V
|
0.4mM
|
.2mM
|
2
|
0.33
|