RF-Cloning.org Forum

Hi vsustar,
This could be a tricky one, but maybe for a reason you haven't considered.

First, while your insert is fairly long, I think it should be fine. In my mind, the shared sequence between RFP and GFP will actually be beneficial on both the 5' and 3' end, because it's going to stabilize binding of the megaprimer along that whole stretch of sequence. There is enough insert after the RFP to make a loop and stick back down to the plasmid, although you might want to add another couple bases to the non-RFP side of the primer just to be safe.

What might actually give you grief is the IRES. As I'm sure you know, they form some interesting secondary structure, and I've ran into problems when trying to clone in or near them. All I can really recommend is that you make sure your primers' melting temps are in the mid 60s, and keep your annealing step hot. All that said, I have successfully cloned an IRES into pEGFP before, and then plunked a >1kb insert down right beside it. I say go for it!

Feel free to send me a private message with a link to your project before firing it off for synthesis. I can probably give a little more feedback if I see everything together in the main interface.
Take care,
-Steve

That's great! Congrats.

Hey Tamjeed,
Are your plasmids really that tiny? If you've only included a portion of the destination plasmid, make sure you are running the extension step for an appropriate amount of time. The primers themselves seem to be a bit unbalanced in terms of their annealing temps, which you should probably try to get within a 1-4°C of one another, but they don't seem to suffer from terrible self-priming at least.
What were your exact PCR conditions (both reaction mix and cycling)?
-Steve

Oh man, that's the coolest. What concentration of each megaprimer and the plasmid did you use (in case someone else wants to try something similar)? Any other pertinent details that might be helpful?
Thanks Gal for letting us know, and congrats.
-Steve

Oh, good find. I had seen the citation to my paper by this pub, but didn't look that closely at it.

"...we describe simultaneous RF cloning of two genes at distinct positions within the expression vector pACYCDuet-1 (Figs. 1 and 2 ), whereas in the second example (Fig. 3 ) the focus is on tandem multicomponents assembly into the expression vector pET21a."

So ya, looks like you can totally mash up whatever you like! They also have a lot of good pointers in the 'Notes' section. I think I'll add a link to this in the Q & A.

Hey Alexander,
I hope your experiment goes well!
The DpnI is crucial, because it chews up all the parental plasmid in the reaction. If you skip the DpnI step you'll end up with tons of transformants, but most of them will have no insert.
Also, you MUST grow up the plasmid in a dam+ strain of E. coli for DpnI to work (the standard sub-cloning strains, like DH5-α, generally fulfill this requirement).
-Steve

Hey Jan,
It's a great question. The larger the plasmid, the lower the final efficiency of the reaction, but 10K is not unreasonable. I've personally used RF-cloning for 13K plasmids. The important thing is that you use a polymerase with very high processivity; in other words, an enzyme which does not fall off the guide strand easily. It's the reason I recommend using iProof and Phusion (they are different brand names of the same enzyme), because they have an extra DNA binding domain that gives them a big boost in processivity.
Unless your plasmid is obnoxiously large, I would say 'give it a try'.
Take care,
-Steve

Hey Dan,
Thanks for the kind words! It's really encouraging when I hear from people that are using the site.
To your questions/comments:
Yes, the predicted time is based on Phusion/iProof (50 bases/sec). If you've been having better success with longer extensions, then of course, keep doing it! It's been a number of years since I settled on that particular value, and things just sort of worked out for me, so I don't have a nice collection of projects of varying size at varying times. I wonder if anyone else out there has played around with different extension times?
That's great info regarding the megaprimer conc. Would you be willing to share the details of one of your optimization experiments along with the check gel? I'd be more then happy to add it to the Q&A.
Take care,
-Steve

Thanks for PMing me the project IDs.
I can see right away why you're not having a lot of success. The plasmid portion of the primers you've designed are extremely high GC, and when I plugged one of them into OligoAnalyzer, it gave me a nasty (-33 kcal/mole) dimer. I'm looking at the plasmid you're using, it seems like you're cloning right into the (admittedly sparse) MCS, so is there a technical reason you've chosen not to use the BamHI and XhoI sites?
For your S._suis_ project, I tried shifting the insert sites to 5388-5391, which should retain your reading frame and not break the his-tag or thrombin site. The primers look really good in those positions, and I'm guessing you could do something similar with the remaining constructs. I'm not sure you're going to be able to make your current primers work unfortunately, they are really ugly, and you could spend a lot of time before you have success :S