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 Primer and promoter 
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Joined: Fri Oct 08, 2010 6:08 am
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Post Primer and promoter
Hi guys,

I was designing a synthetic sequence on Gene Designer. I was wondering if the promoter is located before (5'-3') or after the primer.
In fact, are promoters actually needed in a synthetic sequence?


Sun Nov 14, 2010 11:55 pm
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Joined: Thu Aug 19, 2010 3:18 am
Posts: 26
Post Re: Primer and promoter
There seems to be some confusion here. Basic molecular genetics.

Promoter sequences are regions of DNA where transcription factors bind to promote transcription of the coding sequence. A promoter is needed for gene expression.

Primers are short oligos that bind to complementary sequences. DNA polymerase needs double stranded DNA for initiation. Primers are used in the PCR reaction to amplify a specific section of DNA.

What exactly are you trying to design? What will the synthetic sequence be used for? Its impossible to say whether or not a promoter is required without more specifics.


Mon Nov 15, 2010 12:34 am
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Post Re: Primer and promoter
Thanks for the clarification on primers and promoters. I am designing a gfp sequence for a science project and I didn't know whether the promoter was already in the "origin" which I copy-pasted onto Gene Designer. If not, how do I go about generating or finding one? My gene is 714 bp, 246 a's, 138 c's, 140 g's, and 190 t's.
I found a primer for GFP on Invitrogen and I also found a reverse primer. Should I put the primer and the beginning of the sequence (i.e. before my initial ATG) or at a different place?
Thanks again.


Mon Nov 15, 2010 11:10 pm
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Joined: Thu Aug 19, 2010 3:18 am
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Post Re: Primer and promoter
Lets take a step back even further for a moment. What do you want to do with the GFP? If you want to express the GFP in an organism, then you need a promoter. Remember, promoters are sequences where other proteins that control transcription bind. In order to make RNA copies of the gene, which is necessary to make protein, you need to have transcription. For this, you need a promoter.

Questions you need to ask yourself.

1) What organism do I want to express the GFP in? Different promoters work in different organisms. It matters whether or not you are putting it into bacteria, yeast, plants, animals. It also matters what species it is. I'm going to be honest, at your level, do not attempt plants or animals. To even do plants or animals you first have to work with bacteria.

2) Do you just want to make GFP? GFP is used by biologists as a marker. For example, if I fuse GFP to a specific protein, I can see where in the organism and where in the cell, that protein is located. I can also express just the GFP under specific promoters that are turned on only in certain tissues (like an eye or heart) or under certain conditions (like stress). Unless you have a specific goal, for a beginner who just wants to learn this stuff, I would not attempt to fuse the GFP to another protein. This would require a lot more work. If you just want to see the GFP, then it really doesn't matter. You can make it be on all the time. So here is something to consider. There are promoters in bacteria and yeast that are turned on when exposed to certain nutrients. In this example you could have bacteria with the GFP gene, that aren't expressing it. You then add the nutrient to the cell culture and the bacteria start making the GFP.

Think in detail about those two issues. Be specific about the organism and pattern expression. Then start doing searches for things like "[the organism] promoters [tissue/conditions] expression." This may require skimming a lot of literature to get the answer you need...thats research.

In designing primers, you have to know a few details.

1) What plasmid vector you will be putting the GFP in. This will be determined by the organism, promoter, and several other details.

2) What restriction site you will clone into. This is dependent upon what restriction sites are in the vector, which ones are in the GFP, and what is commercially available.

In designing primers, you can start at the ATG. You need to designe the primers so that at least 18 bps overlap the beginning or end of the sequence. For cloning into a vector you will have to add the appropriate restriction sites before and after the sequence that overlaps the gene. There are other thermodynamic properties of the primer that you need to consider. Tm, self-annealing, hairpin formation, etc. Google primer design and primer design programs, this will give you more information and programs that will aid the design primers.

Do you know anything about PCR?

Cloning genes is routine, but not easy. Its still one of those areas that experienced researchers can get hung up with for a couple of months. Also consider your expenses here and capabilities. PCR can be tricky, requires specific enzymes and buffers. Cloning can be even trickier.


Tue Nov 16, 2010 6:36 pm
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Post Re: Primer and promoter
I was planning on doing a simple transformation of E.coli with GFP. This is going to be my first real experiment and I wanted to start with something simple. I would probably be using the p-T7-SNAP vector that is available through http://www.dna20.com. I know how PCR works, but I don't know how to do it myself (or for that matter, where to get TAQ polymerase or a thermocycler from) so I was planning on ordering the plasmids with GFP already cloned into them for starters, and concentrating on the transformation of E.coli. How do you think I should go about finding which restriction site I am using?

I understand that this is difficult and I thank you for answering my questions.


Wed Nov 17, 2010 4:55 pm
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Joined: Thu Aug 19, 2010 3:18 am
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Post Re: Primer and promoter
Excellent, I like your approach. Your starting from the right angle here with simple experiments in an easy to use organism. I don't have time at the moment to really say much more, but I'm going to take a look at the dna20.com site and see if I can't give you any more advice.


Wed Nov 17, 2010 5:10 pm
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