RF-Cloning.org Forum

erin.green

Member

Registered: 2014-07-01

Posts: 4

RF-cloning to do mutagenesis?

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!

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: RF-cloning to do mutagenesis?

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

EH

Member

Registered: 2015-01-30

Posts: 3

Re: RF-cloning to do mutagenesis?

I saw this post and didn't want to start a new question.  I have a gene that I cloned which ended up with two single mutations about 200-300 bp apart.  i was planing on making a megaprimer (i guess it is actually a large quick change primer about 200bp) of the wild type (without the mutations) for this region of the gene and use it in the 2nd pcr.  I feel this should work no problem... any thoughts?

Thanks-

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: RF-cloning to do mutagenesis?

Hi EH,
Ya, this should work really well, because you'll be complementary across the entire 200-300 (minus 2) region. I'll be surprised if this project gives you any problems at all.
Shoot me a private message with your project id if you want me to have a quick look at it before you send off for primers.
-Steve

EH

Member

Registered: 2015-01-30

Posts: 3

Re: RF-cloning to do mutagenesis?

Thanks Steve,

Primers have been ordered already..had to wait through a snow storm to get them.  First round successful (making megaprimer) starting the second round right now.

Thanks again,

EH

EH

Member

Registered: 2015-01-30

Posts: 3

Re: RF-cloning to do mutagenesis?

Everything worked well and sequence was correct.  Thanks for the help Steve!

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: RF-cloning to do mutagenesis?

Great! I love success stories