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I'm hoping to stick a short sequence (containing a flag tag, stop codon, and restriction site) into a vector I've already constructed. Is there any way I can adapt the RF-cloning protocol to do this? I realize this is a form of Quick-Change PCR, but I've never really had much success using their protocols. Thanks!
Hi Steve,
Just wanted to give you an update. I tried the reactions again with two modifications: 1. After reexamining gels that I ran following the PCR clean-up step, it looked like low levels primers or primer-dimer molecules were still present even after column purification, which may have messed with the secondary PCR. So I re-amplified my inserts and gel purified them prior to the secondary PCR step. 2. I also tried using a new stock of Dpn-i (was worried that our working stock had potentially gone bad). Not sure which modification did the trick, but got TONS of positives the second time around. Thanks for your help!
-Erin
Hi Steve,
I haven't tried plating on the double drugs, but could definitely try that fix. The 3kb construct unfortunately doesn't have a drug marker, so I'm still going to have to try to optimize that one. I did only get modest yields from the initial PCR/cleanup for both megaprimers I've been working on. Both products were around 50ng/uL after PCR purification, so I had to add a good bit of megaprimer to the secondary PCR reactions in order to achieve the concentrations that the site recommended.
The mastermix I used for the secondary PCR contained:
4uL 5x Phusion buffer
0.4uL dNTP mix
0.2uL Phusion
0.5uL vector (diluted to ~150ng/uL)
either 5uL megaprimer (for the 1kb insert, which was at about 50ng/uL) or 15uL megaprimer (for the 3kb insert, which was also about 50ng/uL)
and water to make the final volume 20uL
I cycled the secondary PCR using the 2-step protocol, as follows
1. 98dC for 30s (1x)
2. 98dC for 8s
3. 72dC for 3min
4. Go to step 2 and repeat 15x
5. 72dC for 5min
Let me know what you think!
-Erin
I just learned of rf-cloning from a colleague last week and decided to give it a try (thanks to your very useful website!). Unfortunately, though I followed your protocol and used the recommended insert and vector concentrations in the second PCR (using the 2-step cycling protocol), things don't seem to be working. I recovered a bunch of colonies following my transformation, but upon screening about 50 by PCR for each construct I'm trying to make (currently trying to make two different plasmids), none contained any insert. Any ideas about what might be going wrong?
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