PCR protocols
Note: This is a quick-start protocol. It does not provide the full information about each enzyme. Please refer to the online manual for detailed information about individual enzymes.
Note: To determine the volume of your PCR reactions, 20 ul is normally used when the PCR product purification step is not necessary. For example: colony PCR. 50 ul is used when you need to purify your PCR product.
Go Taq Green
Go Taq green is great for applications that do not required high fidelity of the DNA Polymerase. It’s good for colony PCR, or as a control when your PCR reaction doesn’t work. Also, it comes with a dye. So no need to add DNA loading dye after your PCR reaction.
Recipe:
Template: x ul
100 uM Primer 1: 0.4 ul
100 uM primer 2: 0.4 ul
2X Go Taq Green Master Mix: 10 ul
ddH2O: to make it 20 ul
Total volume: 20 ul
Note: For beginners, pipetting 0.4 ul liquid is challenging. Instead, dilute 100 uM primer ten folds to 10 uM in a new 1.5 ml tube and add 4 ul of each primer to the reaction. Keep the 10 uM primer at -20C for future use.
Thermo cycle:
95°C: 30 sec to 3 min (If you use whole E. coli cell as the template, do 3 min denaturing)
95°C: 30 sec
54°C: 30 sec (the annealing temperature depends on your primer Tm)
72°C: 1kb/min
repeat from step 2, 35 cycles total
72°C: 10 min
12°C: forever
Phusion
Phusion enzyme is a high-fidelity DNA polymerase. It’s great for amplifying genes for cloning. Phusion is very fast too. However, I found Phusion is not as robust as Taq, especially if your PCR product exceeds 2 kb.
Recipe:
Template: x ul
100 uM Primer 1: 0.4 ul
100 uM primer 2: 0.4 ul
5x HiFi Buffer: 10 ul
10 mM dNTP: 1 ul
Phusion Enzyme: 0.5 uL
ddH2O: to make it 50 ul
Total volume: 50 ul
Note: For beginners, pipetting 0.4 ul liquid is challenging. Instead, dilute 100 uM primer ten folds to 10 uM in a new 1.5 ml tube and add 4 ul of each primer to the reaction. Keep the 10 uM primer at -20C for future use.
Thermo cycle:
98°C: 30 sec
98°C: 10 sec
56°C: 30 sec (the annealing temperature depends on your primer Tm+ 3°C)
72°C: 1 kb/15 sec
repeat from step 2, 35 cycles total
72°C: 10 min
12°C: forever
Note: Phusion also comes with a high GC buffer. If you encounter problems with PCR, especially for high GC template and large PCR product, try GC buffer or adding 3 ul DMSO.
Platinum Taq
Platinum Taq is also a high-fidelity DNA polymerase. I found it more robust than Phusion, but it is slower too. It extends DNA at 68°C instead of 72°C.
Recipe:
Template: x ul
100 uM Primer 1: 0.4 ul
100 uM primer 2: 0.4 ul
10x Pt Taq buffer (labeled 10x HiFi buffer): 5 ul
50 mM MgSO4: 2 ul
Platinum Taq: 0.3 ul
10 mM dNTP: 1 ul
ddH2O: to make it 50 ul
Total volume: 50 ul
Note: For beginners, pipetting 0.4 ul liquid is challenging. Instead, dilute 100 uM primer ten folds to 10 uM in a new 1.5 ml tube and add 4 ul of each primer to the reaction. Keep the 10 uM primer at -20C for future use.
Thermo cycle:
94°C: 30 sec-3 min (If you use whole E. coli cell as the template, do 3 min denaturing)
94°C: 30 sec
54°C: 30 sec (the annealing temperature depends on your primer Tm)
68°C: 1 kb/min
repeat from step 2, 35 cycles total
68°C: 10 min
12°C: forever
Note: For longer PCR products: Try adding 3 ul DMSO if your PCR didn’t work for longer products.
Zhen
12/19/17