RF-Cloning.org Forum

Shubhangivirkar

Member

Registered: 2022-11-14

Posts: 2

Trouble for primary PCR

Hi I am Shubhangi,
I have been doing primary PCR for two fragments which is about 4kb and 65bp and not able to get the band for both the fragments
I have tried doing around 2943 bp fragment which came successfully
3 times I tried with different cDNA and same conditions
For the 4kb fragment I have been using the following conditions:
Denaturation- 98 for 30 seconds
                     98 for 8 seconds
Annealing -    55 for 20 seconds
Extension -    72 for 4 minutes
                    72 for 10 minutes

For the 65bp product, I tried to use reduced number of cycles and later increased also, and also tried doing primer dilution but the band didnt come
Following conditions I used for 65bp product:
Denaturation- 98 for 30 seconds
                      98 for 8 seconds
Annealing-     55 for 20 seconds
Extension-     72 for 30 seconds
                     72 for 5 minutes
Please let me know what more changes I can do
Thank you.