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#1 2014-11-24 19:43:44

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!

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#2 2014-11-24 20:44:52

Steve Bond
Administrator
Registered: 2014-01-23
Posts: 112

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

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#3 2015-01-30 15:58:40

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-

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#4 2015-01-30 16:20:37

Steve Bond
Administrator
Registered: 2014-01-23
Posts: 112

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

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#5 2015-02-04 22:17:56

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

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#6 2015-02-18 17:05:38

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!

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#7 2015-02-18 17:16:07

Steve Bond
Administrator
Registered: 2014-01-23
Posts: 112

Re: RF-cloning to do mutagenesis?

Great! I love success stories smile

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