RF-Cloning.org Forum

Hmm, might be a bit difficult. Check out the following method by Peleg and Unger: doi 10.1007/978-1-62703-764-8_6
(PM me if you run into the paywall, I'll send the pdf)
I haven't tried it myself, so I can't really advise you much more than this. Please let us know though if you have any success (or failure).
-Steve

Hey Erin,
This should be a pretty straight forward RF project. I assume you can synthesize the megaprimer directly given how short all the bits are, so just stick it in the website and get the PCR params. It really is just a large quick-change at this point though, because you don't need the full primary PCR reaction.
What are some of the details from your previous bad luck with QC kits?
-Steve

Hey Gabriele,
I think the difference is that the exponential Megaprimer method leads to completely linear vector which needs to be blunt end ligated, while the more standard version of RF-Cloning gives you a circularized, albeit nicked, plasmid. The transformed cells will repair the nicks all on their own, but are just going to degrade the linear DNA; hence the phosphorylation/T4-ligase step. I'm just skimming through the 2014 article by Peleg and Unger again (doi: 10.1007/978-1-62703-764-8_6) and they are not using the exponential method, which is probably why you aren't seeing reference to a ligation step.
Hope this clarifies!
Good luck, and it would be awesome if you share your experience with the exponential method after you've given it a whirl.
-Steve

Hey again Zac,
Before you do anything else, redo the 2° PCR and only use 1 μl of the reaction in the transformation. You've made a common mistake by assuming 'more is better', but you're changing the salt concentration of the comp cells and significantly reducing their efficiency. You should not exceed 5% of the total cell volume when adding DNA, 10% max. The cells are actually sensitive to total DNA as well (20-50 ng is a common target for high efficiency), and there is probably 10-100X too much total DNA (albeit chewed up by DpnI) in the full PCR reaction.
One other thing I've noticed is that you appear to be cloning into the MCS, and there are some really ugly dimers on the plasmid side of both primers. Bump up your annealing temp to 68°C, and you might want to increase your extension time to 3:30 min.
Good luck, and let us know how it turns out
-Steve

Hey Zac,
I think I was able to find your project from the backend, and on first glance everything looks good. The plasmid is a bit big, but by no means horrible. What were the details of your transformation?
-Steve

ps. I've linked the project I think was yours to your account. Go into the project management dashboard and see if it's right.

Hey Sand,
You're probably more or less on track regarding the first bit, but instead of thinking of it as a nicked plasmid, just look at is as a single linear daughter product. When the reverse complement mega-primer binds, it's 3' end is situated right at the terminus of the strand, so it can't initiate any further synthesis. Thus the linear expansion in rf-cloning vs. exponential of normal PCR.
That little + red thingy is just trying to convey that the reverse complement of the plasmid was still synthesized from a parental source by the other half of the mega-primer, and when you combine the two daughter strands, you get a full plasmid with a pair of nicks. They should have changed the colour of the incoming daughter strand to green or something, because it really looks like the parental plasmid acquired a nick somehow (it doesn't).
Does that clarify, or am I just muddying the water further?
-Steve

Any of the main commercial kits should be okay, although I have often suffered from low recovery as well. I've used GeneCleanII mostly (it's a glass milk kit). Your best bet is to fiddle with the primary PCR a bit, and get better amplification if possible. Load everything!

Hey Jan,
The amount of megaprimer to use is provided in the output as 'insert', and is expressed as 'ng' instead of a μM concentration.
-Steve

Have you been plating the entire transformation reaction, or tried transforming ultra-comp cells?
-Steve

You sure can!
When you use the website to design your project, place the '!' points on either side of the sequence you want removed.
If you want, I can have a look at your project before you order the primers.
-Steve

Wonderful! Congrats.
-Steve

You didn't use TE buffer to elute your megaprimer by any chance, did you? The EDTA would mess things up, although I think most PCR cleanup kits have you elute in water or EB. A good positive control would be to set up some random PCR reaction you know works well, and add in the same volume of megaprimer you used in the 2ndary PCR to see if it bungs up the reaction (assuming you have enough megaprimer to mess around with it like that). Obviously you'll also need the test product to be different size than the megaprimer.
Your conditions look fine to me. You might want to try the 3-step protocol, using an annealing temp of 63-65°C for 30-sec. You could also try some PCR additives (handle_url_tag($matches[1])).
One other thing you can try, especially for the 3KB project, is to cut the amount of DNA going into the PCR by 5X (i.e.,about 150ng insert, 13ng plasmid).
Fortunately, you have some 1-cutter restriction sites where you want your inserts, so you can always switch to In-Fusion if the RF-Cloning proves too troublesome.
Good luck!
-Steve