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Dear all,
a rookie question: to me it seems that one of the pitfalls of this technique is the fact that you use a PCR to generate the novel plasmid (with the insert).
Now I am wondering: what is the max size if a plasmid that can be used?
I can imagine it being very hard to use this techniques with plasmids ranging between 6000bps and 10.000 bps (or bigger)? Or is this not a problem?
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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
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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
I see.
And you used some sort of specific protocol?
Or just a common PCR protocol for long templates?
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Hi again Jan,
Sorry for not replying right away, I didn't see your new message until just now.
If you go with the recommended reaction conditions for your particular polymerase, you'll probably be okay, BUT! On your secondary PCR, cut the number of cycles down to 16-18. More cycles will reduce efficiency further.
-Steve
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Hi again Jan,
Sorry for not replying right away, I didn't see your new message until just now.
If you go with the recommended reaction conditions for your particular polymerase, you'll probably be okay, BUT! On your secondary PCR, cut the number of cycles down to 16-18. More cycles will reduce efficiency further.
-Steve
I am going to try it.
I'll use Q5 polymerase, it is supposed to be even better than phusion or is Q5 not the best idea?
What about the reaction settings?
First I'll need to figure out how it works in detail, but I am pretty much going to use it for a plasmid of about 9000bps and 41% GC
The goal is to introduce GFP in the plasmid.
I'll check the website and the tool and if I run into troubles, I'll come back here to ask for more questions.
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Hey Jan,
I've not used Q5 polymerase myself, but from what I can see its very similar to Phusion. It still uses the Sso7d processivity enhancing domain, so I assume it's either a minor tweek to the original Phusion enzyme, or maybe they've grabbed a polymerase out of some other thermo-tolerant bug. I don't see any reason not to use it, and look forward to hearing about your experience. Just make sure to follow NEBs directions regarding annealing temps.
Your plasmid is also pretty big, so you might want to use some high comp cells if a first attempt comes up blank.
Best of luck!
-Steve
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