|
Nuclear Region HS Takara PCR Optimization
Test 4.0ul of 5.0uM Primer Concentration
| HS Takara RXN Mixture 3.0ul Instagene
|
conc
|
1X
|
5.5
|
'
|
| Milli Q water |
|
0.1 |
0.55 |
|
| Takara HS Buffer |
5X |
2 |
11 |
|
| dNTP |
2.5uM |
0.8 |
4.4 |
|
| Primer Mix |
5uM |
4 |
|
|
| HS Taq |
5U |
0.1 |
0.55 |
|
| Template |
Instagene |
3 |
16.5 |
|
|
|
|
33 |
6.0ul / RXN
|
|
|
|
|
add 4.0ul Primer
|
| A PCR cycle |
|
|
|
|
| 98C |
10 sec |
|
|
|
| 60C |
1 min |
|
|
|
| x35 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
| AMP Lane |
Template |
Primer |
Amt of Template |
|
| 1 |
3 Instagene |
B1 |
3 |
|
| 2 |
3 Instagene |
C1 |
3 |
|
| 3 |
3 Instagene |
D1 |
3 |
|
| 4 |
3 Instagene |
E1 |
3 |
|
| 5 |
3 Instagene |
F1 |
3 |
|
| 6 |
4 Instagene |
B1 |
3 |
|
| 7 |
4 Instagene |
C1 |
3 |
|
| 8 |
4 Instagene |
D1 |
3 |
|
| 9 |
4 Instagene |
E1 |
3 |
|
| 10 |
4 Instagene |
F1 |
3 |
|
| 11 |
AV 1ng |
B1 |
1 |
|
| 12 |
AV 1ng |
C1 |
1 |
|
| 13 |
AV 1ng |
D1 |
1 |
|
| 14 |
AV 1ng |
E1 |
1 |
|
| 15 |
AV 1ng |
F1 |
1 |
|
|
|
|
|
|
| *AV 2ng Positive Control |
|
|
|
|
Load 2.0ul Sample
AV 2ng - bad dilution
Nuclear Region HS Takara PCR Optimization
Compare 1.0ul of 20uM Primer vs 4.0ul of 5uM (old primer dilution)
| HS Takara RXN Mixture
|
conc
|
1X
|
3
|
'
|
| Milli Q water |
|
0.1 |
0.3 |
|
| Takara HS Buffer |
5X |
2 |
6 |
|
| dNTP |
2.5uM |
0.8 |
2.4 |
|
| Primer Mix |
5uM |
4 |
|
|
| HS Taq |
5U |
0.1 |
0.3 |
|
| Template |
Instagene |
3 |
9 |
|
|
|
|
18 |
6.0ul / RXN
|
|
|
|
|
add 4.0ul Primer
|
| HS Takara RXN Mixture |
conc |
1X |
3 |
|
| Milli Q water |
|
3.1 |
9.3 |
|
| Takara HS Buffer |
5X |
2 |
6 |
|
| dNTP |
2.5uM |
0.8 |
2.4 |
|
| Primer Mix |
20uM |
1 |
|
|
| HS Taq |
5U |
0.1 |
0.3 |
|
| Template |
Instagene |
3 |
9 |
|
|
|
|
27 |
9.0ul / RXN
|
|
|
|
|
add 1.0ul Primer
|
| A PCR cycle |
|
|
|
|
| 98C |
10 sec |
|
|
|
| 60C |
1 min |
|
|
|
| x35 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
| AMP Lane |
Template |
Primer |
Amt of Template |
Primer
|
| 1 |
3 Instagene |
F2 |
3 |
4.0ul of 5uM
|
| 2 |
4 Instagene |
F2 |
3 |
4.0ul of 5uM
|
| 3 |
AV 20ng |
F2 |
1 |
4.0ul of 5uM
|
| 4 |
3 Instagene |
F2 |
3 |
1.0ul of 20uM
|
| 5 |
4 Instagene |
F2 |
3 |
1.0ul of 20uM
|
| 6 |
AV 20ng |
F2 |
1 |
1.0ul of 20uM
|
|
|
|
|
|
| *AV 20ng Positive Control |
|
|
|
|
Load 2.0ul Sample
well 2 has a weak band
|
Alien genes
