|
Polymerase Chain Reaction
First
Stage
The standard 1st
stage PCR mix contains the following:
|
|
1
tube |
10
tubes |
20
tubes |
|
10*PCR
buffer |
5 |
50 |
100 |
|
dNTP
|
0.5 |
5 |
10 |
|
Sense Primer |
0.25 |
2.5 |
5 |
|
Anti-Sense Primer |
0.25 |
2.5 |
5 |
|
Water |
38.8 |
388 |
774 |
|
Taq
Polymerase |
0.2 |
2.0 |
4.0 |
|
Total |
45 |
450 |
900 |
1.
Make a mastermix and
then aliquot into 45
ml
volumes
2.
Add 5
ml
of HCV cDNA to the mix
3.
Overlay with 50
ml
of liquid paraffin
2nd
Stage
The standard 2nd
PCR mix contains the following:
|
|
1
tube |
10
tubes |
20
tubes |
|
10*PCR
buffer |
2 |
20 |
40 |
|
dNTP
|
0.2 |
2 |
4 |
|
Sense Primer |
0.1 |
1 |
2 |
|
Anti-Sense Primer |
0.1 |
1 |
2 |
|
Water |
17.5 |
175 |
350 |
|
Taq
Polymerase |
0.08 |
0.8 |
1.6 |
|
Total |
20 |
200 |
400 |
1.
Make a mastermix and
then alliquot into 20
ml
volumes
2.
Add 2
ml
of 1o product to the mix
3.
Overlay with 50
ml
of liquid paraffin
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