RF-Cloning.org Forum

Manu

Member

Registered: 2020-09-30

Posts: 2

Second PCR not working

Hi,

I am trying to clone two 3-4kb sequences into a 7.5kb plasmid and am having some trouble getting the second PCR to work. I get abundant and high purity first PCR product. I am using a KAPA high fidelity PCR mix from Roche with the following cycling conditions:
    Initial denaturation: 95°C 3min
    Denaturation: 98°C 20s   
    Annealing: 65°C 15s              25 cycles
    Extension: 72°C    12min    
    Final extension: 72°C    12min
I also tried a couple variations: reducing the annealing temp to 60 + increasing cycles to 35 or decreasing cycles to 15 + 65 annealing temp + 9min elongation time. I either get no colonies or very few which turn out to be empty backbone. I found that increasing the amount of DpnI from 1ul to 1.5ul eliminates empty plasmid colonies and instead I get no colonies at all. Any help would be greatly appreciated.
Project IDs:
https://www.rf-cloning.org/rf_cloning_project.php?proj_id=437b59a76752a5b82ea2b7ac912be788
https://www.rf-cloning.org/rf_cloning_project.php?proj_id=620503d22897aaf36b010296f50ee554

Thanks!

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: Second PCR not working

Hi Manu,
Your inserts are on the large side. Not to say this isn't doable, but the efficiency is going to be low. My suggestion would be to use InFusion, if possible, otherwise to just make sure you're using highly competent cells, using an appropriate amount of DNA in the transformation, gently centrifuging the cells down and plating everything. You might even want to do 3 or 4 transformations.
For the cycling conditions, you should never go over 15 cycles on the secondary. It causes artefacts to accumulate and further reduces the efficiency. The final extension  can be reduced as well.
Stick with the 65°C anneal and a 5 minute extension. I am not familiar with the KAPA polymerase personally but a brief bit of Googling suggests to me it's a good choice.
I'll keep my fingers crossed for you,
-Steve

Manu

Member

Registered: 2020-09-30

Posts: 2

Re: Second PCR not working

Hi Steve,

Firstly, thanks a lot for your prompt reply. I reduced the cycles to 15, and tried either 5min or 7min elongation time. I also spun down the cells and plated everything as you suggested, using high competency bacteria (I always do a transformation control with empty vector to check). Still I get no colonies:( If you have any further tips or ideas for things to change in the reaction conditions, I would really appreciate it. 

Thank you.

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: Second PCR not working

Hi Manu,
Ya, I'm not sure what more I can recommend. Have you tried smaller inserts previously and had success? This one is tricky, so if it's your first attempt... Again, I highly recommend In-Fusion. The primers you have are still good and it's way higher efficiency than the RF-cloning secondary PCR.
Sorry for not being much help
-Steve

linh_

Member

Registered: 2021-01-15

Posts: 3

Re: Second PCR not working

Hi Manu,

Maybe it's a little late but in case somebody has the same question.
I think the problem is your insert is too large. I also have to insert the same side with yours and couldn't succeed, so I divided my insert into 1,5kb and try RF cloning one by one insert. It takes more step and more works, but it has more chance to succeed, I think.

Good luck.