ligation - very small insert - problems during ligation (May/28/2008 )

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QUOTE (killerkoz17 @ Jun 3 2008, 11:43 AM)

QUOTE (popdok @ Jun 2 2008, 07:17 PM)

thanks for advice

but I would rather dephosphorylate my digested vector than buy expensive phosphorylated oligos

That didn't make sense, but i understand you dont want to buy the phosphorylated oligos (really not that expensive). Any way, dephosphorylating the vector must be done to prevent background colonies. I would phosphorylate your oligos or ds insert using T4 PNK to get the phosphates for ligation.

I think i see how what Pernese is talking about will work. No phosphates present anywhere and relying on the attraction between the sticky end bases to hold the insert in the vector and then letting the bacteria to repair the nicks? Is that right Pernese?

Swanny, I was referring to phosphates on the DNA required for ligation, so i dont think the ATP in the buffer has anything to do with what i was saying.

Sorry, missed that thought.

The idea of not having ligated ends and getting the cells to do the work is the basis of LIC and SLIC cloning, but they rely on ~15-20 bp complementary sequences. I think you'll come a cropper against the thermodynamics of a 4bp overhang if you just do standard RE-site cloning without at least some phosphates.

Thinking of the phosphorylation reaction, the latest edition of Sambrook has good protocols for adapting oligos and cloning.

-swanny-

QUOTE (popdok @ May 28 2008, 06:42 AM)

I digested plasmid pHannibal with 2 different restr. enz., that gives me 3kb fragment with 1 blunt and 1 stick end. My insert is very small (about 67bp) and is made by 2 ss oligonucleotides hybridization (syntetized as primers; after hybrydization I obtain ds oligonucleotide with appropriate ends - the same ends will occur after digestion). The problem is during ligation this small insert into digested fragment of my plasmid. Did anyone do similar ligation? I tried several times with diffirent molecular ratio, and still without results. Is it possible to ligate so small fragment? What do you suggest?
thanks for any helpful answers

I'm doing similar cloning now. my insert is 45bp +RE sites
I've made a very stupid mistake that I think I'll never forget: the insert/vector ratio should be mole ratio!!! 55555555555555555555
I diluted my insert by 10000fold

I'm checking my colonies now.

-rosepolaris-

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