RF-Cloning.org Forum

Shoot. The DpnI-free control just gives you an idea as to the upper limit of colonies you could expect. Assuming you're going to lose 99% of the parental plasmid in the process, if you only see a couple hundred colonies from the positive control, then that would be an indication you are going to struggle to get anything from the DpnI treated sample.
Regardless, did you try any of the suggestions in the new post I linked to? There were some interesting ideas there.

Hi Sybr,
The small insert will help, but that is a rather large plasmid.
A few bits of info and advice:
- Don't expect to see much by running a gel on the secondary PCR. No band means nothing.
- You're running the secondary PCR for too many cycles. Cut it back to 15.
- Your extension time in the secondary is long. Google isn't immediately telling me what the processivity of Q5 is, but NEB asserts it's 'fast', so I'd dial it way back to what the RF-Cloning tool is suggesting (4:18 min extension)
- Do as many transformations as you deem appropriate from the volume of secondary PCR you have after the run, and then plate everything (i.e., gentle centrifuge into a soft pellet, resuspend in a reasonable volume, and spread it all on a plate).
- Include a positive control -> take a sample before DpnI treatment and use it to transform. You should see many colonies from that.

These are the easy suggestions I can make based on your project and protocol. I'll cross my fingers they will be enough.
Good luck,
-Steve

Edit: It seems like your timing was most opportune. Please see this new post that includes additional suggested protocol modifications.

You should be able to get away with up to 70°C.  Generally, you want to keep the forward and reverse primers within a few degrees of one another, so I'd also update the reverse primer as appropriate.
Good luck!
-Steve

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

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

Hi jk,
Yes, you should be mixing these two oligos together in equal molar ratio and move straight into the secondary PCR step. I believe I've found your project in the database but it looks like your megaprimer is actually 114 bp? Regardless, if the plasmid I'm seeing is correct (final size 7383 bps), you're looking at setting up a reaction with 27 ng total of your mixed primers and 88 ng of plasmid. Annealing temp 72C, extension time of 2:26 min, 15 cycles. What's the doi on the paper you've pulled these primers from? I'll have a look at their methods.
Give it another try and let me know how it turns out!
-Steve

Hi Asli,
I found three projects attached to your userid, swapping out luciferase for D4, B6, and E3_5.
Your cycling conditions look fine to me, but you are going to have a low efficiency reaction because the plasmid is getting a bit big and the replacement of large fragments usually makes things worse.
Another concern I have is the IRES in between the luciferase and GFP genes. The strong secondary structure can get in the way of proper annealing and extension.
I often encourage people to use In-Fusion, when possible, and I would suggest the same to you. You have a ScaI and NotI pair that will knock out most of the luciferase gene and linearize the parental for the InFusion reaction.
Aside from that, all I can really suggest is poking around more in the forum to see some of the troubleshooting tips people have had through the years. You might have success with some additives.
I wish you the best of luck,
-Steve

Hey Hyla,
I would expect that you have caused a lot of high molecular weight artifact to build up in your reaction with this protocol. Try again, but reduce the extension step down to 140 sec and reduce the number of cycles to 15.
And I didn't mean anything more than what I said about the deletion. If you delete a couple dozen base pairs, it doesn't seem to have much of an affect on overall efficiency, but deleting hundreds or thousands of base pairs does. It's just something to keep in mind.
-Steve

Hi Hank,
I wrote that response too quick, without explaining my rational. The issue with Ranjani's example is that the entire insert would have been included in the hybrid primer. Doing a PCR would probably work wether the insert was present in the plasmid clone or not, and will give the same 'positive' result either way. If you have a long insert, then the hybrid primer can definitely be used for QC because there is more information in the plasmid than is present in the primer. Does that make sense?
-Steve

This is a difficult project because of the size. I'm not sure there is much more I can do for you.
Are you gently spinning down the cells after transformation and plating all of them? Do you have ultra competent cells you can use?
Just to cover all your bases, you can also run a positive control (no DpnI) to make sure everything up to the transformation is going correctly.
If this construct is key to your work you can also hire a company to synthesize it. I think you would be looking at USD$1500-$2000 for a 17Kb plasmid (maybe less if you don't need all of the elements in that plasmid).
One other possible solution is to cut out the region you are working on and move it into a smaller plasmid, do the rf-cloning reaction, and then swap it back into the large plasmid. I'm pretty sure I found your project in the database, and it looks like you have restriction site options around your insert.
I hope this helps.
-Steve

That's great about the positive clones! Congrats.
As for the second issue, I'll need you to clarify something for me. You aren't trying to make two mutations in the same reaction, are you?
Let's pretend you want to make a point mutation at position 100 and another point mutation at 400. To do this, you will first make the mutation at 100, purify the plasmid, confirm with Sanger sequencing, and only then will you start to make the second mutation at 400, using the new plasmid as template.
The cycle conditions you provided look fine to me, but why have you diluted your primers to 0.35 ng/μl (i.e., 5 ng / 14.5 ul)? That seems very low, as I usually have them at 5 ng/μl for a single basepair substitution project like yours. If they are diluted in TE buffer, you may be messing up the pH and Mg++ level a bit.
-Steve

Hi Abdul,
Your destination plasmid is very large, so I'd anticipate you will struggle to get this to work.
How many colonies did you screen? If you are seeing hundreds of colonies on your plate, I would suggest pooling them into groups of 10 to reduce the number of PCR reactions on empty vector colonies. Hopefully you will see one or two pools with the insert, then you can re-screen just that target pool to narrow it down to a clone with your insert.
Is there any chance you can switch to In-Fusion? This will be easier, if you can.
-Steve