1. Gradient PCR using Taq polymerase with 125ng primer concentration
M: 1 kb ladder
1: 60°C
2: 62°C
3: 64.4°C
4:66.8°C
5: 69.2°C
6:71.5°C
284D FP: 1.25ul (125ng)
284D R.P: 1.25ul (l25ng)
Template: 1.5ul (50ng)
PCR premix: 21ul

2. Gradient PCR using Taq polymerase with 5picomole primer concentration
M: 1 kb ladder
1: 63.7°C
2: 65.7°C
3: 67.6°C
4:69.3°C
5: 70.8°C
6:71.9°C
284D FP: 1.72ul (5 picomole/ul)
284D R.P: 1.72ul (5 picomole/ul)
Template: 1.5ul (50ng)
PCR premix: 21ul
T= 95 30s
T= 65 to 72 1min
T= 68 7 min

3. M
M: 1 kb ladder
1: PCR product
2: PCR product without template
3: PCR without primers
284D FP: 1.72ul (5 picomole/ul)
284D R.P: 1.72ul (5 picomole/ul)
Template: 1.5ul (50ng)
PCR premix: 21ul

4. M
M: 1 kb ladder
1: 354 PCR product ( 5 picomole/ul)
2: 354 PCR product (2.5ul primers)
3:284 PCR product (2.5ul primers)
FP: 2.5ul (5 picomole/ul)
R.P: 2.5ul (5 picomole/ul)
Template: 2ul (60ng)
4mM dNTPs: 1.25ul
Pfu- 0.5ul
DMSO:0.75ul
10x buffer: 2.5ul
H20: 14.5ul
T=95 30s
T=68 1min
T=68 6min
18cycles

5. PCR using 3picomole/ul primer concentration- also tried with Emerald PCR master mix
containing Pfu hot start polymerase (Takara) M
M: 1 kb ladder
1: 284 PCR product with emerald ( 3 picomole/ul primer)
2: 354 PCR product with emerald (3 picomole/ul primer)
3:284 PCR product (3ul primers)
4: Template alone
Lane 1 &2
284D FP: 3ul (3picomole/ul)
284D R.P: 3ul (3 picomole/ul)
Template: 1.5ul (50ng)
Emerald PCR premix: 12.5ul
Lane 3
4mM dNTPs: 1.25ul
Pfu-0.5
10x buffer-2.5ul H
T=95 30s
T=68 1min
T=68 6min
18cycles