Cloning

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Cloning

Postby nekameneka » Dec 01 2016 5:29 am

Hi everyone, I am back for help again.
I have basic cloning question and I would like to ask detailed explanation for the future reference.
I have ORF cDNA clone of my gene of interest in the cloning vector (pGEM-T vector). Of course I want to transfer it to make expression plasmid (possibly pEF-BOS).
I guess this should be very simple. In the instructions, manufacturer says the coding sequence can be easily obtained by digesting the vector with proper enzymes and it also can be amplified by PCR.

What I understand is that I can choose an enzyme and cut the vector and the empty plasmid I want, than I can do ligation and transformation in E.coli.
My question is...which enzyme to choose? Can someone explain to me in detail what is important to consider when choosing enzymes (I guess this is very basic so I hope I am not taking too much of your time)? And I will have to do PCR also right?

Thank so much! :)
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Re: Cloning

Postby relaxin » Dec 05 2016 12:46 pm

You need to choose the enzyme(s) whose recognition site(s) are flanking the gene of interest so that your gene can be excised out in one piece. Then you have to find an expression vector with the same enzyme recognition site(s) in the multiple cloning region. It is sometimes difficult to do. The easier way is to copy the gene of interest by PCR using primers with built-in enzyme recognition site(s) that are available on the expression vector.
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Re: Cloning

Postby nekameneka » Dec 06 2016 10:15 pm

Hi, thank you for the reply.
I did not find proper enzymes in the empty vector I have so I will insert restriction sites by annealed oligo cloning instead doing PCR.
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Re: Cloning

Postby relaxin » Dec 07 2016 1:33 pm

Yes, you can use linker/adaptor to clone the gene of interest into the expression vector. You can also do blunt-end ligation of the gene to the vector. Just make sure the blunt-ended vector is dephosphorylated. You also need to play attention to the distance between the ATG codon and rbs, if it is a bacterial expression vector.
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Re: Cloning

Postby nekameneka » Dec 11 2016 10:19 am

Thanks a lot! :)
Here is a new question: which sample buffer is suitable for native page of proteins with pl higher than 8?
I searched a lot but I didn't find some good explanation or recipe...
Thank you!
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Re: Cloning

Postby relaxin » Dec 11 2016 9:23 pm

I seldom do native PAGE, so I let other experts answer your question.
I would try 10 mM sodium acetate pH 5.0.
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Re: Cloning

Postby nekameneka » Dec 11 2016 11:34 pm

Thank you, but isn't the pH 5 too low for protein of 8 pl?
I also found information that sample buffer can be: Tris-glycerol-bromophenol as for basic (6-8) pl proteins but in case of pl higher than 8 I should reverse electrodes, maybe I will try that...
Also I found for pl higher than 8 I should do "blue" native PAGE, which means my sample buffer would be: 50mM Tirs pH 6.8, 50 % glycerol and 0.1% Coomassie blue G-250, but I am not sure is this 1x buffer?! :cry:
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Re: Cloning

Postby mdfenko » Dec 12 2016 8:06 am

blue native page is a method for determining the mw of proteins without using sds. native page separations are driven more by charge than weight.

when you need to run native page of basic proteins then acid page is recommended (and, yes, the electrodes are reversed).

you should be able to find the formulation for acid page in posts in this forum (http://molecularbiology.forums.biotechniques.com/viewtopic.php?f=4&t=42751).
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Re: Cloning

Postby nekameneka » Dec 12 2016 8:53 pm

Hi, thank you for the answer.
I use commercial gels so I don't prepare them by myself. Ok, for the first time I will try acidic buffer and reverse of electrodes. I will let you know how it went and if I had any problem.
I would like to see dimerization of my protein.
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Re: Cloning

Postby mdfenko » Dec 13 2016 7:39 am

a small problem with just replacing the electrode buffer, the buffer in the gel may interfere with the run.

can you prepare your own gels?
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Re: Cloning

Postby Morcone » Feb 16 2017 1:42 am

mdfenko wrote:a small problem with just replacing the electrode buffer, the buffer in the gel may interfere with the run.

can you prepare your own gels?


Yeah, you're right it does run. I guess one needs to create their own gels like you say.
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