HELP, What is kind of System

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gezigezi
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HELP, What is kind of System

Post by gezigezi » Mon Jan 16, 2006 10:34 pm

Hi, I am a newbie, I am just using Alkaline Phosphatase, Calf Intestinal (CIP) to construct the operon. I try many times, but I still got a lot of colonies on my control plasmids. Does someone use CIP and give me some better advice,such as incubate time and incubate temperature using CIP.Thanks for your advice! :D

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canalon
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Post by canalon » Tue Jan 17, 2006 4:57 am

Well if you do not give your protocol it is hard to help trouble shhot what you are doing. But If I understand you are preparing plasmids (no operon) by digesting them, then dephosphorylating them with CIP to avoid self ligation before you add the fragment you want to clone and teh ligase.
Did you check the digestion efficency of your digestion. And what is your control? ARe you using a plasmid with a blue/white system?

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gezigezi
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Here is my protocol.

Post by gezigezi » Tue Jan 17, 2006 3:24 pm

Yes, It is exactly like what you said,for my pUC19 plasmid (contain my gene I), I use Pst I single digestion,then purify DNA by spin-column purification (qiagen), and according to the protocol (NEB) ,Here is my protocol:
sterilized water : 2.5ul
NEbuffer3: 3ul
DNA(vector): 24ul
C.I.P: 0.5ul
incubate 37C for 60min, then purify DNA by spin-column purification.
Then I add another gene II(NsiI/PstI), ligate and transformation ,the control is without the gene II, I found both have the similar colonies,so I do not use blue/white screening system like a-complementary.
Actually, what I want is to ligate several gene in the same plasmid,to construct the operon.
So the trouble is , my control has a lot of self-religation, Could you give me some advice, Appreciate it!

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canalon
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Re: Here is my protocol.

Post by canalon » Tue Jan 17, 2006 4:36 pm

Ok Then, the first thing I would recommand is either to run an a few µl of your plasmid digestion (before or after purif) on a gel to check if the digestion was complete or to run the whole mix in a gel and cut out the band of the correct MW (probably better, there should be no undigested plasmid this way).

For the dephosphorylation step maybe you can increase time or concentration of CIP.

I do not really see what else can be changed.
You can also go on this more technical forum where you may find more ideas. But for me, whenever possible, I definitely prefer to make double digestion with 2 different enzymes when possible, it allow me to control everything including the direction of insertion.
Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)

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