RF-Cloning.org Forum

janhavi23

Member

Registered: 2021-02-13

Posts: 3

Secondary PCR transformation not working

Hello!

My project id is https://www.rf-cloning.org/rf_cloning_project.php?proj_id=970adea87a9e2d1e58c546c5a093315d

I have tried both the 2-step and 3-step PCR method with Phusion polymerase and annealing of 60C for the 3-step method.

Haven't gotten any colonies. I would be grateful for any suggestions!

I am going to try a fresh primary pcr and a lower annealing temperature as of now.

Thank you!

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: Secondary PCR transformation not working

Hello,
Without looking too closely, I see that your target plasmid is fairly large at ~11Kb. Efficiency starts to noticeably decrease above 10Kb. No colonies at least means you aren't fighting false positives right away, so that's a good thing
Working in your favour is that you aren't trying to delete a big chunk of the parental and your insert isn't overly large. Before messing around with different PCR conditions, my first suggestion is to make sure your transformation efficiency is high. Are you running controls there? Are you plating out the entire transformation reaction?
If you're convinced the transformation protocol is perfect, then type out the exact PCR protocols you have tried and we'll see if anything pops out.
-Steve

janhavi23

Member

Registered: 2021-02-13

Posts: 3

Re: Secondary PCR transformation not working

Hi Steve,

Thank you so much. I use XL1 competent cells made in our lab for the transformation. We use them for all our cloning and a positive control transformation usually gives us a lawn growth. For example, the parent plasmid was cloned using golden gate assemble with 100 ng of plasmid and I had several (~50) colonies even with just 10% of the transformation plated. I could borrow comp cells from another lab to try though.
The PCR conditions I am using are exactly as described in the project link-
121 ng Parent plasmid, 150 ng insert
4 uL phusion buffer, 0.4 10 mM dNTPs and 0.2 uL phusion pol made to a total of 20 uL
2-step : 98 30s, 98 8s, 72 3:38 min (15 cycles) and 72 5 min final extension.
3 step : additional annealing at 60 for 20s

janhavi23

Member

Registered: 2021-02-13

Posts: 3

Re: Secondary PCR transformation not working

*I plated the entire transformation reaction on a single plate.

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: Secondary PCR transformation not working

I think you just need to try it a couple more time, to be honest. I'm not seeing any gross dimer issues and if you're following along with recommended cycling conditions, there's not that much more that will improve things. You can attempt to add a little DMSO to the reaction but you aren't fighting a high GC primer, so I don't actually think it will help. If you can get some higher competency cells, that may be the best bet. Something in the 10^9 range if you aren't there already.
Otherwise, i think the best I can do for you is cross my fingers!
Good luck,
-Steve