Gradient PCR using Taq polymerase with 125ng primer concentration

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Polymerase Chain Reaction (PCR)

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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

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

M 1 2 3 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

M 1 2 3 4 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

PCR using 3picomole/ul primer concentration- also tried with Emerald PCR master mix containing Pfu hot start polymerase (Takara) M 1 2 3 4 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 H20-13.25 T=95 30s T=68 1min T=68 6min 18cycles