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Plasmid duplication sites

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Plasmid duplication sites

Postby mehdi71000 » Sat Jun 16, 2007 12:34 pm

Hi every one
i have trouble understanding dna duplication sites for different species. Can you name them for me please.
is salI for ecoli?
what are the others?

Thanks

What plasmid below is to be used in for? And is it poisonous?
cheers
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Postby canalon » Sat Jun 16, 2007 2:27 pm

All those sites are not duplication sites. They are restriction sites. That is were DNA is cut by enzymes that recognize a particular DNA sequence.

Plasmids usually have one Origin (of replication) where the the replication is started. Some of those origins are more or less specific to the bacterial species, and one has to be carefull when transferring plasmids between species that they would be "compatible". Most of the commercialy available plasmid are designed to work in E. coli (but would likely be transferred without tooo much problem in many Enterobactariacea). If you look for Life science catalogs you will find plasmids specially enginered for transformation of plants (usually a derivative of the Ti plasmid from Agrobacterium tumefasciens), mammalian cells, insect cells etc...
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Postby mehdi71000 » Sat Jun 16, 2007 4:05 pm

thanks mate
So if I use ndele and hindilll enzyme on this vector it will be cut and the bgal gene would be separated from ampicllin and origin?
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Postby david23 » Sat Jun 16, 2007 5:55 pm

And you will lose the origin as well, pretty smart of you.
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Postby mehdi71000 » Sat Jun 16, 2007 8:36 pm

Cheers mate need

would it still duplicate or do I still need to add an orgin?

Thanks
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Postby mehdi71000 » Sat Jun 16, 2007 8:55 pm

I’ve heard cells without a nucleus don’t use dna they only use Rna, is this true?
One thing, is the proteins and enzyme responsible in dna duplication in the nucleus or outer nucleus of cells with nucleus?

thanks
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Postby canalon » Sat Jun 16, 2007 9:40 pm

mehdi71000 wrote:thanks mate
So if I use ndele and hindilll enzyme on this vector it will be cut and the bgal gene would be separated from ampicllin and origin?


Yes you will have 2 single linear strand of DNA. And if you do not purify the fragment with the origin and insert soething that would match the sticy end to circularize it agin, it won't be able to be replicated in a cell. On the other hand this is where you would be able to insert a selected fragment of DNA containing a gene of interest to clone it. And If say you inserted there a fragment containing a replication origin adapted for a specific target (other than E. coli, say eucaryotic cell), a selective marker for the same target anf your favorite gene, you would have build a shuttle vector.
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Postby mehdi71000 » Sat Jun 16, 2007 10:03 pm

So to get this to work i need the enzyme to cut it. then add the replication origin of a plant and enzyme to close it? So without purification 50% of the dna would glow in a plant. So I should use the other pvib vector with salI sites.
Any ideas on where to find salI enzyme and eukaryotic cell origins?
thanks
and I’ve heard cells without a nucleus don’t use dna they only use Rna, is this true?
One thing, is the proteins and enzyme responsible in dna duplication in the nucleus or outer nucleus of cells with nucleus?

thanks
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Postby david23 » Sat Jun 16, 2007 11:00 pm

They all use DNA, the cells.

Cells cant survive on RNA alone because

1. RNA is unstable and gets degraded often, which is a normal process in cells

2. Neither bacteria nor the higher up organisms have the kind of RNA polymerase that can replicate RNA from RNA.

Ok now back to you, a nucleus plays a lot of other roles. DNA replication happens inside the nucleus.

And while we are at this, why didnt you buy the eukaryotic plasmid with GFP? You wouldnt need all this cutting and stuff. restriction enzymes arent cheap either.
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Postby mehdi71000 » Sat Jun 16, 2007 11:33 pm

AKHH GFP they cost $250 for 20 ug and $100 shipping to UK.
I found some restriction enzymes on the net, SalI.
Any ideas on where to find some shuttle eukaryotic plasmids with SalI sites.
Oh yeh if I cut the pvib plasmid with SalI how do I attach it to the other dna?


thanks
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Postby david23 » Sun Jun 17, 2007 1:46 am

The gene you are using should also come with some restriction sites, if not then Sal .
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Postby mehdi71000 » Sun Jun 17, 2007 1:40 pm

Ok mate I play with some ecoli first I modify for plant next month. So there is no chance this plasmid can work for eukaryotic cells?
cheers
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