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Hi Steve,
First, thats really a great site and it helps a lot in designing RF.
I'm trying to design primers for inserting 2 genes into pBABe-puro plasmid (one into the MCS and second instead of Puro) .
The trouble is that in both genes the first 21 nucleotides are simmilar.
Do you have any suggestions to overcome it?
Maybe to design a longer primer for the gene?
Thanks
Gal
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Hey Gal,
It probably won't matter. The secondary PCR is going to prime to the plasmid, and the insert sequence is just along for the ride.
I haven't ever tried to include two different mega-primers into a single 2° reaction though, and don't think I recommend it. By all means give it a try, but I predict it's going to fail. Just do them successively, and it should work out okay.
If you do try to do both, and it works, come back to the forum and let us know the details!
Best of luck,
-Steve
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Thanks.
Gal
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Take a look at that:
Application of the Restriction-Free (RF) Cloning
for Multicomponents Assembly
Yoav Peleg and Tamar Unger
(Methods Mol Biol. 2014;1116:73-87)
or at: http://www.ncbi.nlm.nih.gov/pubmed/24395358
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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.
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Steve,
The simultaneous RF cloning work fine. I'v inserted two fragments into pBABE-Puro on the first try (mCherry instead of puromycin and another gene at the MCS).
Gal
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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
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I used the protocol described in: Methods Mol Biol. 2014;1116:73-87 with minor changes.
Give me an Email and I'll send it to you so you'll be able to upload it to the website.
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You can send PMs via the little email links to the left of our comments. Alternatively, my email address can be found in the Q & A on the main page (I'd rather not post it in the forum, where more spam bots like to lurk).
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If anyone is interested in Gal's protocol, I have uploaded the document he sent me here
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