Admin: Created page with "=== Protocol for Phusion
=== *Add the following volumes to a round-lidded PCR tube:
{| cellspacing="1" cellpadding="1" border="1" style="width: 326px; height: 225px..."

2021-02-01T19:40:30Z

Created page with "=== Protocol for Phusion === *Add the following volumes to a round-lidded PCR tube: {| cellspacing="1" cellpadding="1" border="1" style="width: 326px; height: 225px..."

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=== Protocol for Phusion ===

*Add the following volumes to a round-lidded PCR tube: 

{| cellspacing="1" cellpadding="1" border="1" style="width: 326px; height: 225px;"
|-
! scope="col" | Reagents 
! scope="col" | Volume (µL) 
|-
| ddH2O 
| up to 50 µL 
|-
| HF 5x buffer 
| 10 
|-
| ddNTPs 
| 1 
|-
| Primer A 
| 0.5 
|-
| Primer B 
| 0.5 
|-
| DNA 
| X 
|-
| DNA polymerase 
| 0.5-2 
|-
| DMSO (optional) 
| (1.5) 
|-
| Total 
| 50 
|}

Note: GC buffer can be used instead of HF buffer when using GC-rich templates

*Add the tube to the thermocycler and use the following times:

{| cellspacing="1" cellpadding="1" border="1" style="width: 441px; height: 182px;"
|-
! scope="col" | Step
! scope="col" | Temperature (°C)
! scope="col" | Time (s)
! scope="col" | Number of cycles
|-
|
Initial denaturation

| 98
| 30
| 1
|-
| Denaturation
| 98
| 5-10
| 35-45
|-
| Annealing
|
>20nt; Tm + 3°C

<20nt; Tm (lower of the two primers)

|
10-30

(time depends on complexity)

| 35-45
|-
| Extension
| 72
| 15-30/kb
| 35-45
|-
| Final extension
| 72
| 5 min
| 1
|-
| Infinite hold
| 4
| hold
| 1
|}

PCR - Revision history

Admin: Created page with "=== Protocol for Phusion === *Add the following volumes to a round-lidded PCR tube: {| cellspacing="1" cellpadding="1" border="1" style="width: 326px; height: 225px..."

Admin: Created page with "=== Protocol for Phusion
=== *Add the following volumes to a round-lidded PCR tube:
{| cellspacing="1" cellpadding="1" border="1" style="width: 326px; height: 225px..."