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PCRed on restriction sites -- how do I purify it?

subcloning pcr

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#1 n00b

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Posted 27 February 2012 - 10:03 AM

Hello. I'm very new to subcloning, so please bear with me.

I have a gene (KCNE2) currently in the mammalian vector, pci-neo. I need to remove it from pci-neo and put it into the bacterial expression vector pet28a. The pet28a has an insert that can be liberated with NheI and HindIII.

Someone in the lab previously put KCNE1-pci-neo into pet28a by PCRing on NheI and HindIII sites onto either end of the KCNE1 insert (unfortunately they aren't here anymore and they didn't take notes, so I can't follow precisely what they did). I'm going to try to do that as well.

I ordered primers, and am running the PCR with the primers to add NheI and HindIII flanking KCNE2 now. My question is: what next? After the PCR, do I PCR purify the result, and then can I go straight to cutting it? Or do I need to run the PCR result on a gel, gel purify, and then cut it?

For my vector, I know I need to cut it, run a gel, gel purify, treat with CIP, and then add with the cut insert in varying ratios. But I'm not sure what to do with the result of my PCR.

Thank you very much for any help you're able to provide!

#2 bob1

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Posted 27 February 2012 - 11:51 AM

Check out the activity of your restriction enzymes in PCR buffer - if it is less than about 70%, you will be better off making a bulk prep of the PCR and cleaning it up using ethanol precipitation or a PCR clean up kit. Note that you may have to do a sequential digest of the PCR product, which would mean that you would need to clean it up between reactions.

If the fragement that you are cutting out of the plasmid is less than about 20 bp, you can also purify these using a PCR clean up kit, as most of these won't retain small fragments. As you are probably using non-compatible ends, you probably don't need to de-phosphorylate the plasmid, but it won't hurt to do so. Note that CIP is quite hard to deactivate, so you may be better off using one of the other de-phosphorylases.

Also note that the ratios are molar ratios, not just mass - use this formula: insert mass in ng = ratio x (insert size in bp/vector size in bp) x vector mass.

#3 n00b

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Posted 27 February 2012 - 12:15 PM

Thank you for your response, Bob.

Are you saying that I can add the restriction enzymes themselves to the PCR (which I'm running to add the requisite restriction sites to the insert?) So instead of PCRing to add sites, then clean up, then cutting, I can PCR to add sites & cut at the same time, then clean up, then go straight to ligation?

Thanks!

#4 bob1

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Posted 27 February 2012 - 12:28 PM

Sort of. Sometimes REs will work in PCR buffer so you can run your PCR (and presuming it is a single band product), then just do the digest by adding restriction enzymes straight to the reaction mix and incubating at the appropriate temperature (usually 37 deg C).

#5 n00b

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Posted 27 February 2012 - 01:47 PM

Alright, I'll try this! Thanks for your help.

#6 bob1

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Posted 27 February 2012 - 03:15 PM

Check the compatability of the enzymes with the PCR buffer first - it depends on where you get your enzymes from as to the advice given, so you should check the supplier's manual.

#7 phage434

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Posted 28 February 2012 - 05:12 AM

I rarely disagree with Bob1, but here I have to. In your PCR reaction all of the components are present to extend the 3' end of DNA fragments. If you cut your DNA with the RE, leaving a 5' overhang (this happens with most enzymes) then the PCR enzymes will blunt the end of the cut. For diagnostic purposes, such as cutting and observing gels, this is fine, but for cloning, it destroys the overhangs.

PCR reactions used prior to cloning must be cleaned up prior to RE cutting.

Also, no one has mentioned the classic error of making PCR primers with RE sites, but neglecting to add additional 5' bases to allow the RE to cut. Add junk bases 5' of your cut site -- I like to add 6.

#8 bob1

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Posted 28 February 2012 - 11:37 AM

I have found that most polymerases, but especially Taq, can be heat killed by heating the PCR to 95 for more than 20 minutes, which should stop any further action. However, phage's advice is good (and it seems he does a lot more cloning than I do), especially the last sentence!

#9 n00b

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Posted 26 March 2012 - 02:47 PM

Thanks for both of your help! I transformed successfully today. Posted Image





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