RF-Cloning.org Forum

Sybr_Green

Member

Registered: 2022-01-14

Posts: 3

First PCR went OK, second PCR does not work.

Hi!
I'm currently working of this project
https://www.rf-cloning.org/rf_cloning_project.php?proj_id=6b352888bd47334817381af6a2e47602
I can get the first PCR to work and extract the band from gel, but the secondary PCR fails to yeld any succesful colony (actually any colony at all).

For the first PCR I follow this protocol:

ID 98°C 30s

D 98°C 8s
A 62°C 20s    x35
E 72°C 5s

FE 72°C 5min

The 62°C annealing temp is calculated on the annealing portion of the primer to the portion of plasmid containing the framgent that I want to insert using the Tm calculator of NEB (I'm using the NEB Phusion HotStart Flex).

For the second PCR I follow this protocol, using 35ng of insert and102 ng of plasmid as recommended by your online tool

ID 98°C 30s

D 98°C 8s
A 68°C 20s            x15
E 72°C 2min50s

FE 72°C 5min

Again the 68°C annealing temp is calculated on the annealing portion of the megaprimer to the portion of plasmid where I want to insert the fragment using the Tm calculator of NEB.

I also tried a 2step one

ID 98°C 30s

D 98°C 8s
                             x15
E 72°C 2min50s

FE 72°C 5min

Both of them are not working, what would you suggest?